Hapten-carrier conjugates and uses thereof

ABSTRACT

The present invention provides compositions comprising a conjugate of a hapten with a carrier in an ordered and repetitive array, and methods of making such compositions. The conjugates and compositions of the invention may comprise a variety of haptens, including hormones, toxins and drugs, especially drugs of addiction such as nicotine. Compositions and conjugates of the invention are useful for inducing immune responses against haptens, which can use useful in a variety of therapeutic, prophylactic and diagnostic regimens. In certain embodiments, immune responses generated using the conjugates, compositions and methods of the present invention are useful to prevent or treat addiction to drugs of abuse and the resultant diseases associated with drug addiction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/125,402, filed May 10, 2005; which is a divisional of U.S. Pat. No.6,932,971, issued Aug. 23, 2005; and claims benefit of the filing dateof U.S. Provisional Application No. 60/396,575, filed Jul. 18, 2002; thedisclosures of each of which are entirely incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is in the fields of medicine, public health,immunology, molecular biology and virology.

2. Related Art

Addictive drug abuse disorders carry with them a number of specific,well recognized sequelae that have both societal and economicconsequences. These include death, disease, violence, crime, loss ofemployment, reduced productivity, relationship and familial breakdown,and the spread of HIV and other sexually transmitted diseases. Theeconomic cost to United States society from drug abuse (excludingtobacco) was an estimated $98 billion in 1992, the last year for whichreliable data are available (“The economic costs of alcohol and drugabuse in the United States-1992”, National Institute on Drug Abuse).These costs include crime ($59.1 billion), premature death ($14.6billion), impaired productivity/workplace accidents ($14.2 billion),welfare ($10.4 billion), health care ($5.5 billion), and motor vehicleaccidents. These costs are borne primarily by government (46%), drugabusers and their families (44%). It is well recognized that drug abuseremains a serious problem in society. Three years after the 1992 study,in 1995, NIDA estimated drug abuse costs to the society was $110billion.

The per se use of drugs of abuse can have deleterious effects on theuser. However, it is recognized that the addictive nature of these drugsare both central to the problems associated with such drug use, andunderlie the inability to treat both addicted individuals and reduce theprevalence of drug addiction in the society.

The most widely used addictive drug in the world is tobacco. Nicotine,an alkaloid derived from tobacco leaves, is the principal addictivecomponent of tobacco. In 1999, 46.5 million adults in the United Stateswere current smokers. Cigarette smoking is the single leading cause ofpreventable death in the United States. According to the Centers forDisease Control and Prevention (CDC) 430,000 annual deaths areattributable to cigarette smoking in the United States. Lung cancer,coronary heart disease, chronic lung disease, and stroke are the maincauses of death. Smoking is not only dangerous to individuals, it alsoresults in staggering societal costs. The estimated smoking-attributablecost for medical care in 1993 was more than $50 billion and the cost oflost productivity and forfeited earnings due to smoking-relateddisability was estimated at $47 billion per year. Thus, the totaleconomic cost associated with nicotine addiction is greater than thecombined costs for all other types of addictive drugs.

Despite recent advances in behavioral and pharmacologic treatments, thevast majority of cigarette smokers who try to quit will fail (foroverview see Fiore et al. (2000) Treating tobacco use and dependence,clinical practice guideline, US Department of Health and Human Services,Public Health Service). Nicotine replacement therapy is one currentlyused medication, either in the form of nicotine gum, inhaler, nasalspray or transdermal patches. The efficacy of transdermal nicotinepatches alone has been questioned in a placebo-controlled,double-blinded clinical trial (Joseph et al., N. Engl. J. Med. (1999),340:1157-1158; Jorenby et al., N. Engl. J. Med. (1999) 340:685-691).Furthermore, adverse effects of nicotine gum such as mouth irritation,sore jaw muscles, dyspepsia, nausea, hiccups and paresthesia anditching, erythema, sleep disturbances, gastrointestinal problems,somnolence, nervousness, dizziness and sweating for the nicotine patchwere observed. A treatment with the antidepressant bupropion canincrease the abstinence rates at 12 months to about 30% (Jorenby et al.,supra).

Novel approaches to the treatment and prevention of addiction, tonicotine and to other drugs, are clearly needed. Immunization strategiesto modify the behavioral effects of drugs have been the subject ofinvestigation since 1974. Both active immunization withmorphine-6-hemisuccinate-BSA and passive immunization with the resultantantibodies reduced heroin self administration in rhesus monkeys (Bonese,et al. Nature 252:708-710 (1974); Killian, et al Pharmacol. Biochem.Behav. 9:347-352 (1978).) Immunization has also proven effective againstcocaine addiction. Active immunization reduced the effect of subsequentcocaine administration in rats (Carrera et al Nature 379:727-730 (1995),and both active and passive immunization was demonstrated to abolishself administration (Fox et al. Nature Med 2:1129-1132 (1996)). Morerecently, immunization with GNC-KLH conjugate abolished selfadministration in cocaine-addicted rats (Carrera et al Proc. Nat. AcadSci USA 97:6202-62061992 (2000)) and both immunization with GND-KLHconjugate or transfer of anti-cocaine monoclonal antibodies blockedcocaine effects (Carrera et al Proc. Nat. Acad Sci USA 98:1988-1992(2001).

Antibodies have been raised against phencyclidine (PCP) and showeffectiveness in reducing PCP levels in the brain, reducing behavioraleffects, and show similar abilities to block the physiologic effects ofPCP analogs (Hardin et al. J Pharmacol Exp Ther 285:1113-1122 (1998);Proksch et al. J. Pharmacol Exp Ther. 292:831-837 (2000)). Antibodieshave also been successfully raised against methamphetamine in rats(Byrnes-Blake et al. Int Immunopharmacol 1:329-338 (2001)). U.S. Pat.No. 5,256,409 discloses a vaccine comprising a carrier protein bound toone hapten from the desipramine/imipramine class of drugs and anotherhapten from the nortriptyline/amitriptyline class of drugs.

Therefore, immune responses can be raised against drugs, the antibodiescan block drug action, and animal models have demonstrated thatvaccination is effective as a general approach to the treatment of drugabuse and addiction. It is believed that generating an immune responseshould block the actions of the drug by preventing it from entering thecentral nervous system (Carrera et al Nature 379:727-730 (1995). Byreducing the rewards associated with drug use, the addicted individualis no longer motivated to consume the drug.

As the addictive effect of the drugs is caused by their action in thebrain, antibodies in serum should be able to reduce drug delivery tobrain. Cerny (WO 92/03163) described a vaccine and immunoserum for useagainst drugs of abuse. The vaccine consisted of a hapten bound to acarrier protein. Also disclosed therein was the production of antibodiesagainst drugs, and the use of these antibodies in the detoxification ofone who has taken the drug.

Nicotine, cocaine, heroin and most drugs of abuse are haptens, which arenot immunogenic. Coupling of haptens to protein carriers typicallyenhances their immunogenicity.

Several different nicotine haptens, carriers and methods of couplinghave been described. Matsushita et al. (Biochem. Biophys. Res. Comm.(1974) 57, 1006-1010) and Castro et al. (Eur. J. Biochem. (1980) 104,331-340) prepared nicotine haptens conjugated to bovine serum albumin(BSA) via a linker at the 6-position of the nicotine. Elsewhere, Castroet al. (Biochem. Biophys. Res. Commun. (1975) 67, 583-589) disclosed twonicotine albumin conjugates: N-succinyl-6-amino-(±)-nicotine-BSA and6-(sigma-aminocapramido)-(±)-nicotine-BSA. Noguchi et al. (Biochem.Biophys. Res. Comm. (1978) 83, 83-86) prepared a nicotine-BSA conjugatewith nicotine conjugated at the 1-position of the nicotine. Langone etal. (Biochemistry (1973) 12, 5025-5030 and Meth. Enzymol. (1982) 84,628-640) prepared the hapten derivativeO-succinyl-3′-hydroxymethyl-nicotine and conjugated it to bovine serumalbumin and keyhole limpet hemocyanin. According to the procedures ofLangone et al. (supra), Abad et al. (Anal. Chem. (1993) 65, 3227-3231)synthesized the nicotine hapten 3′-(hydroxymethyl)-nicotinehemisuccinate and coupled it to bovine serum albumin for immunization ofmice to produce monoclonal antibodies to nicotine. Isomura et al. (J.Org. Chem. (2001) 66, 4115-4121) provided methods to synthesize nicotineconjugates linked to the 1′-position of nicotine, which were coupled tokeyhole limpet hemocyanin (KLH) and BSA. The conjugate to KLH was usedto immunize mice and to produce monoclonal antibodies against nicotine.Svensson et al. (WO 99/61054) disclosed nicotine-haptens conjugated viathe pyridine ring and further disclosed a nicotine-hapten conjugated toKLH and the induction of nicotine-specific IgG antibodies using suchconjugates. When administered in the presence of complete Freund'sadjuvant, nicotine-specific ELISA titres of 1:3000 to 1:15500 weremeasured, while in the absence of Freund's adjuvant titres of 1:500 to1:3000 were detected. Ennifar et al. (U.S. Pat. No. 6,232,082) disclosednicotine haptens coupled via the pyrrolidine ring and disclosed anicotine-hapten conjugated to recombinant Psuedomonas aeruginosaexotoxin A (rEPA) and the induction of nicotine-specific IgG antibodieswhen the conjugates were administered in the presence of completeFreund's adjuvant. Swain et al. (U.S. Pat. No. 5,876,727) disclosed thecoupling of a nicotine hapten to BSA and the induction ofnicotine-specific IgG antibodies in mice when the conjugates were givenin a mixture with complete Freund's adjuvant.

The feasability of a vaccination against nicotine has been shown inprinciple (Hieda et al., J. Pharm. Exp. Ther. (1997) 283, 1076-1081;Hieda et al., Psychopharm. (1999), 143, 150-157; Hieda et al., Int. J.Immunopharm. (2000) 22, 809-819; Pentel et al., Pharm. Biochem. Behav.(2000), 65, 191-198, Malin et al, Pharm. Biochem. Behav. (2001), 68,87-92). Covalent conjugates of nicotine with KLH or rEPA were producedand injected into mice or rats in the presence of complete Freund'sadjuvant, and induced nicotine-specific IgG antibodies. Vaccine efficacywas demonstrated by several different ways. After challenge withnicotine, more nicotine remained bound in serum and nicotineconcentrations were lower in the brain in the nicotine-KLH ornicotine-rEPA immunized groups of rats compared to the control groupimmunized with carrier alone. Immunization also reduced thepsychopharmacological activity associated with nicotine, as immunizedanimals were also less susceptible to the effect of nicotine onlocomoter activity, dopamine release (Svensson et al. WO 99/61054) andrelief of nicotine withdrawal symptoms.

The above art demonstrates the efficacy of vaccine compositionscontaining complete Freund's adjuvant to induce immune responses againstnicotine. Complete Freund's adjuvant is one of the most potentsadjuvants available, however because of its side effects its use is notapproved for humans. Therefore, there exists a need for vaccinecompositions able to induce strong immune responses against nicotinewithout the use of complete Freund's adjuvant. Further, while BSA hasbeen used successfully as a carrier in animal models it may not beappropriate for use in human vaccine compositions because of the risk ofadverse reactions such as the risk of transmitting prion disease(variant Creutzfeldt-Jakob disease). A further challenge to thedevelopment of an effective vaccine against nicotine is the need for animmune response able to rapidly decrease nicotine available forabsorption by the brain. Nicotine from cigarettes is taken up by mucosalsurfaces especially in the mouth and lungs and transported via the bloodto the brain. If nicotine-specific antibodies are to be successful inreducing nicotine delivery to brain, they will have to overcome the veryhigh arterial nicotine concentration that is presented to brain withinseconds of inhalation (Hieda et al., 1999, supra). Therefore, highconcentrations of nicotine-specific antibodies in the blood, which aremainly of the IgG subtype are needed. In mucosal surfaces IgA antibodiesare the primary subtype. Accordingly, in addition to the antibodies inblood, nicotine-specific antibodies of the IgA subtype in the lung wouldbe beneficial for neutralizing nicotine inhaled during smoking before itbegins circulating in the blood.

Cholera toxin, a known carrier protein in the art, can induce IgAantibodies, in particular after intranasal administration. Cholera toxincan also act as an adjuvant, eliminating the need for complete Freund'sadjuvant in a vaccine composition. However, when cholera toxin isadministered as a mucosal adjuvant it stimulates a predominantlyT_(H)2-type immune response with increased interleukin-4 levels andassociated increments in total and specific IgE antibody levels(Yamamoto et al., (1997) Proc. Natl. Acad. Sci USA 94, 5267-5272). Afternasal immunization in the presence of cholera toxin, IgE-associatedinflammatory reactions developed within the lungs of mice (Simecka etal., (2000) Infect. Immunol. 68, 672-679, Hodge et al., (2001) Infect.Immunol., 69, 2328-2338). Despite the promise of intranasal immunizationin the presence of cholera toxin, there is also the potential to developadverse immunopathological reactions characterized by pulmonary airwayinflammation (Hodge et al., (2001) Infect. Immunol., 69, 2328-2338).

Therefore, there exists a need for carrier systems able to stimulateimmune responses against hapten without the use of toxic adjuvants,without the use of poorly tolerated carrier proteins and, in certainsituations, without stimulation of potentially pathologic T_(H)2 immuneresponses. Novel carrier systems meeting these specifications can beused towards the formation of novel conjugates and compositions suitablefor the treatment of addiction, among other conditions, for which thereis currently an urgent need.

BRIEF SUMMARY OF THE INVENTION

We have found that haptens attached to core particles leading to highlyordered and repetitive hapten arrays are surprisingly effective ininducing immune responses, particularly antibodies, against haptens.Core particles, containing a first attachment site, and haptens,containing a second attachment site, are linked through said first andsecond attachment sites to form said ordered and repetitive haptenarrays. The interaction between first and second sites may be direct, ormay involve at least one other molecule, e.g. a linker.

In one embodiment, the first attachment site naturally occurs in thecore particle. Alternatively, the first attachment site is added bychemical coupling or by recombinant techniques. Preferred firstattachment sites comprise amino groups, carboxyl groups or sulfhydrylgroups. Preferred amino acids comprising a first attachment site areselected from lysine, arginine, cysteine, aspartate, glutamate tyrosineand histidine. Particularly preferred are lysine residues.

Suitable second attachment sites on haptens are amine, amide, carboxyland sulfhydryl groups. There is a wide range of compounds that have beendeveloped to enable crosslinking of peptides/proteins or conjugation ofprotein to derivatized molecules, by forming a covalent bond with areactive group of a protein molecule of the core particle.

Core particles with a first attachment site of the invention include anyparticle suitable for the formation of ordered repetitive arrays. Insome embodiments such core particles include virus-like particles(VLPs), bacteriophage, bacteriophage virus like particles, pili, and thelike. In certain embodiments these are HbcAg VLPs, bacteriophage VLP andtype I pili. The invention also provides variant forms of the coreparticles that remain able to form ordered repetitive structure. Variantforms include recombinant and natural forms, and mutant forms of coreparticles. In certain embodiments, the mutant forms of the core particleinclude those where the type of first attachment site, or number of saidsites, differ from the parent. Alteration of the number of lysineresidues on the core particle are particularly preferred.

In certain embodiments, conjugates of the invention comprise haptenssuitable for inducing immune responses against a variety of molecules,including but not limited to toxins, hormones and drugs. More preferredare drugs, and yet more preferred are drugs of abuse or addictive drugs.Haptens of the invention contain a second attachment site for linkage tothe first attachment site of the core particle, either directly or viaat least one linking molecule. In one embodiment, the hapten is suitablefor inducing immune responses against cocaine, for example succinylatednorcocaine.

Preferred embodiments of the invention are nicotine-hapten conjugates.Nicotine haptens suitable for the conjugates of the present inventioncan have at least one, preferably one, side chain bonded to any positionon either the pyridine or the pyrrolidine ring of the nicotine. Thoseskilled in the art know how to produce suitable derivatives of nicotinehaptens. For example, nicotine may be chemically derivatized at the 3′position to provide an hydroxyl residue that is suitable for reactionwith reagents such as succinic anhydride to formO-succinyl-3′-hydroxymethyl-nicotine. This nicotine derivative may becoupled to amino acids of the core particle, such as lysine, using theactivation reagent EDC. In a further preferred embodiment theO-succinyl-3′-hydroxymethyl-nicotine can be activated with EDC and theresulting activated carboxylic group is stabilized byN-hydroxysuccinimide. In other embodiments, haptens are produced byacylation of nornicotine with succinic anhydride in methylene chloridein the presence of two equivalents of diisopropylethylamine. Such anicotine hapten is then coupled to core particles of present inventionwith an activating reagent e.g. HATU. Other methods and processes forsynthesizing haptens suitable for conjugates and compositions or theinvention are provided.

The present invention provides compositions comprising a core particleand a hapten, suitable for use in inducing immune responses.Compositions of the invention include vaccine compositions, with orwithout additional pharmaceutically acceptible excipients or adjuvants.Methods for immunization are provided. More preferred is intranasalimmunization.

Compositions of the invention induce immune responses, including theproduction of antibodies. Therefore, in another embodiment, theinvention provides methods of producing said antibodies against suchhaptens. Such antibodies of the invention are useful in treatment orprevention of diseases and for the detection of haptens, for example inthe methods of diagnosing diseases or diseases associated with thepresence of one or more haptens in the tissues or circulation of ananimal.

In a related embodiment, the invention is useful for the prevention ortreatment of diseases, disorders or conditions which include, but arenot limited to, poisoning by toxins, disregulation of hormone levels,drug intoxication, or drug addiction and the like. Immunization with thehapten-carrier conjugates of the invention results in an immune responseagainst the hapten, such that immune molecules, particularly antibodies,bind the hapten. Passive transfer of antibodies is also useful for thetreatment and prevention of diseases. Treatment of addiction is alsouseful in the treatment of other diseases and conditions associated withaddiction.

We have found that nicotine-hapten conjugates attached to virus-likeparticles induce high nicotine-specific IgG antibodies. The presentinvention therefore provides a therapeutic for nicotine addiction, whichis based on an ordered and repetitive VLP-nicotine conjugate. Thistherapeutic is able to induce high titers of anti-nicotine antibodies ina vaccinated animal. High antibody titers are induced even in theabsence of adjuvants and encompass not only IgG but also IgA subtypes.Furthermore, this therapeutic is, surprisingly, not associated withinduction of potentially pathologic immune responses such asinflammation. Therapeutic compositions of the invention comprise atleast one nicotine hapten molecule and a VLP, or at least one nicotinehapten and an alternative core particle such as HbcAg or pili.

Thus, the invention embodies methods of treatment and preventioncomprising the use of the conjugates and compositions of the invention.Such methods are useful in the therapy and prophylaxis of diseases,disorders and conditions associated with drugs, hormones and toxins.

In a further embodiment of the invention, a pharmaceutical compositionis provided for treating nicotine addiction, palliating nicotinewithdrawal symptoms, facilitating smoking cessation or preventingrelapse comprising a therapeutically effective combination of thevaccine composition of the invention and an additional agent. In oneembodiment, the additional agent is selected from the group consistingof: anti-depressant; nicotine receptor modulator; cannabinoid receptorantagonist; opioid receptor antagonist; monoamine oxidase inhibitor;anxiolytic or any combination of these agents.

Other embodiments of the invention are kits suitable for diagnosis andscreening that utilize the conjugates, compositions and methods of thepresent invention. Other embodiments of the present invention will beapparent to one of ordinary skill in light of what is known in the art,the following drawings and description of the invention, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B depict SDS-PAGE and Westernblot analysis of Nic-Qβconjugates. The nicotine derivate Suc-Nic was coupled to Qβ at differentconcentrations (1×, 5×, 50×, 100×, and 500× molar excess). Aliquots ofthe reaction solutions were loaded on a 16% SDS-PAGE gel and stainedwith Coomassie Blue (A). From a gel run in parallel, proteins weretransferred onto nitrocellulose and detected with an antiserum raisedagainst nicotine-cholera toxin followed by a HRPO-conjugated goatanti-mouse IgG and ECL detection (B). Molecular weight markers are givenon the left margin.

FIG. 2 depicts Nicotine-specific IgG antibodies and IgG titer. Sera fromvaccinated mice were tested for reactivity against nicotine coupled toBSA by ELISA. Optical densities at 450 nm obtained for each serumdilution are shown (A). Titers were calculated from the dilution thatgives half-maximal optical density (B). Average of three mice in eachgroup are shown.

FIGS. 3A, 3B, 3C and 3D depict Nicotine-specific IgG subtypes. Sera fromvaccinated mice were tested for reactivity against nicotine coupled toBSA by ELISA and detected with secondary antibodies specific for IgGsubtypes IgG1 (A), IgG2a (B), IgG2b (C) and IgG3 (D). Optical densitiesat 450 nm obtained for each serum dilution are shown. Average of threemice in each group are shown.

FIG. 4 depicts Nicotine-specific IgE antibodies. Sera from vaccinatedmice were tested for reactivity against nicotine coupled to BSA by ELISAand detected with secondary antibodies specific for the IgE subtype.Optical densities at 450 nm obtained for each serum dilution are shown.Average of three mice in each group are shown.

FIG. 5 depicts Nicotine-specific IgA antibodies. Sera from vaccinatedmice were tested for reactivity against nicotine coupled to BSA by ELISAand detected with secondary antibodies specific for the IgA subtype.Optical densities at 450 nm obtained for each serum dilution are shown.Average of three mice in each group are shown.

FIGS. 6A and B depict the efficacy of the Nicotine-VLP vaccination.

Mice were immunized with Nicotine-VLP and concentrations of nicotine inserum and brain were measured after injection of 3H-nicotine. Averagesof four or five mice per group are shown.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are intended to provide further explanation of the invention asclaimed.

Definitions

The following definitions are summaries of concepts commonly understoodby one of ordinary skill in the relevant art and are provided for thepurposes of comprehension of the following invention but are not meantto be a limitation of the invention.

Adjuvant: The term “adjuvant” as used herein refers to non-specificstimulators of the immune response or substances that allow generationof a depot in the host which when combined with the vaccine andpharmaceutical composition, respectively, of the present invention mayprovide for an even more enhanced immune response. A variety ofadjuvants can be used. Examples include complete and incomplete Freund'sadjuvant, aluminum hydroxide and modified muramyldipeptide. Furtheradjuvants are mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, andpotentially useful human adjuvants such as BCG (bacille Calmette-Guerin)and Corynebacterium parvum. Such adjuvants are also well known in theart. Further adjuvants that can be administered with the compositions ofthe invention include, but are not limited to, Monophosphoryl lipidimmunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts(Alum), MF-59, OM-174, OM-197, OM-294, and Virosomal adjuvanttechnology. The adjuvants can also comprise a mixture of thesesubstances.

Immunologically active saponin fractions having adjuvant activityderived from the bark of the South American tree Quillaja SaponariaMolina are known in the art. For example QS21, also known as QA21, is anHplc purified fraction from the Quillaja Saponaria Molina tree and it'smethod of its production is disclosed (as QA21) in U.S. Pat. No.5,057,540. Quillaja saponin has also been disclosed as an adjuvant byScott et al, Int. Archs. Allergy Appl. Immun., 1985, 77, 409.Monosphoryl lipid A and derivatives thereof are known in the art. Apreferred derivative is 3 de-o-acylated monophosphoryl lipid A. Furtherpreferred adjuvants are described in WO00/00462, the disclosure of whichis herein incorporated by reference.

However, an advantageous feature of the present invention is the highimmunogenicty of the inventive compositions. As already outlined hereinor will become apparent as this specification proceeds, vaccines andpharmaceutical compositions devoid of adjuvants are provided, in furtheralternative or preferred embodiments, leading to vaccines andpharmaceutical compositions for treating drug addiction, preferablynicotine addiction, being devoid of adjuvants and, thus, having asuperior safety profile since adjuvants may cause side-effects. The term“devoid” as used herein in the context of vaccines and pharmaceuticalcompositions for treating drug addiction, preferably nicotine addiction,refers to vaccines and pharmaceutical compositions that are used withoutadjuvants.

Animal: As used herein, the term “animal” is meant to include, forexample, humans, sheep, elks, deer, mule deer, minks, mammals, monkeys,horses, cattle, pigs, goats, dogs, cats, rats, mice, birds, chicken,reptiles, fish, insects and arachnids.

Antibody: As used herein, the term “antibody” refers to molecules whichare capable of binding an epitope or antigenic determinant. The term ismeant to include whole antibodies and antigen-binding fragments thereof,including single-chain antibodies. Most preferably the antibodies arehuman antigen binding antibody fragments and include, but are notlimited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv),single-chain antibodies, disulfide-linked Fvs (sdFv) and fragmentscomprising either a V_(L) or V_(H) domain. The antibodies can be fromany animal origin including birds and mammals. Preferably, theantibodies are mammalian e.g. human, murine, rabbit, goat, guinea pig,camel, horse and the like, or other suitable animals e.g. chicken. Asused herein, “human” antibodies include antibodies having the amino acidsequence of a human immunoglobulin and include antibodies isolated fromhuman immunoglobulin libraries or from animals transgenic for one ormore human immunoglobulins and that do not express endogenousimmunoglobulins, as described, for example, in U.S. Pat. No. 5,939,598,the disclosure of which is incorporated herein by reference in itsentirety.

Active immunization: As used herein, the term “active immunization”refers to the induction of an immune response in an individual,typically an animal, elicited by the administration of an immunogen,vaccine, antigen or hapten-carrier conjugate. By contrast, passiveimmunization refers to the conferral of immunity in an individual by thetransfer of immune molecules or cells into said individual.

Alphavirus: As used herein, the term “alphavirus” refers to any of theRNA viruses included within the genus Alphavirus. Descriptions of themembers of this genus are contained in Strauss and Strauss, Microbiol.Rev., 58:491-562 (1994). Examples of alphaviruses include Aura virus,Bebaru virus, Cabassou virus, Chikungunya virus, Easter equineencephalomyelitis virus, Fort morgan virus, Getah virus, Kyzylagachvirus, Mayoaro virus, Middleburg virus, Mucambo virus, Ndumu virus,Pixuna virus, Tonate virus, Triniti virus, Una virus, Western equineencephalomyelitis virus, Whataroa virus, Sindbis virus (SIN), Semlikiforest virus (SFV), Venezuelan equine encephalomyelitis virus (VEE), andRoss River virus.

Antigen: As used herein, the term “antigen” refers to a molecule capableof being bound by an antibody. An antigen is additionally capable ofbeing recognized by the immune system and/or being capable of inducing ahumoral immune response and/or cellular immune response leading to theactivation of B- and/or T-lymphocytes. This may, however, require that,at least in certain cases, the antigen contains or is linked to a Thcell epitope and is given in adjuvant. An antigen can have one or moreepitopes (B- and/or T-cell epitopes). The specific reaction referred toabove is meant to indicate that the antigen will preferably react,typically in a highly selective manner, with its corresponding antibodyor TCR and not with the multitude of other antibodies or TCRs which maybe evoked by other antigens. Antigens as used herein may also bemixtures of several individual antigens.

Antigenic determinant: As used herein, the term “antigenic determinant”is meant to refer to that portion of an antigen that is specificallyrecognized by either B- or T-lymphocytes. B-lymphocytes responding toantigenic determinants produce antibodies, whereas T-lymphocytes respondto antigenic determinants by proliferation and establishment of effectorfunctions critical for the mediation of cellular and/or humoralimmunity.

Association: As used herein, the term “association” as it applies to thefirst and second attachment sites, refers to the binding of the firstand second attachment sites that is preferably by way of at least onenon-peptide bond. The nature of the association may be covalent, ionic,hydrophobic, polar or any combination thereof, preferably the nature ofthe association is covalent.

Attachment Site, First: As used herein, the phrase “first attachmentsite” refers to an element of the core particle to which the secondattachment site located on the antigen or antigenic determinant mayassociate. The first attachment site may be a protein, a polypeptide, anamino acid, a peptide, a sugar, a polynucleotide, a natural or syntheticpolymer, a secondary metabolite or compound (biotin, fluorescein,retinol, digoxigenin, metal ions, phenylmethylsulfonylfluoride), or acombination thereof, or a chemically reactive group thereof. Multiplefirst attachment sites are present on the surface of the non-naturalmolecular scaffold in a repetitive configuration.

Attachment Site, Second: As used herein, the phrase “second attachmentsite” refers to an element associated with the hapten to which the firstattachment site on the surface of the non-natural molecular scaffold mayassociate. The second attachment site of the hapten comprises anychemical moiety, preferably a amine, an amide, a carboxyl, a sulfhydryl,hydroxyl, aldehyde, acylhalogenide, hydrazine, diazonium, or hydrazide,or further chemical moieties able to specifically react with the firstattachment site. Moreover, the second attachment site may comprise apolypeptide, a peptide, a sugar, a polynucleotide, a natural orsynthetic polymer, a secondary metabolite or compound (biotin,fluorescein, retinol, digoxigenin, metal ions,phenylmethylsulfonylfluoride), a combination thereof, or a chemicallyreactive group thereof. At least one second attachment site is presenton the hapten. The term “hapten” with at least one second attachmentsite” refers, therefore, to a hapten construct comprising at least thehapten and the second attachment site. However, in particular for asecond attachment site, which is not naturally occurring within thehapten, these haptens comprise a linker which associates the hapten withthe second attachment site, or more preferably, already comprises orcontains the second attachment site.

Bound: As used herein, the term “bound” refers to binding or attachmentthat may be covalent, e.g., by chemically coupling, or non-covalent,e.g., ionic interactions, hydrophobic interactions, hydrogen bonds, etc.Covalent bonds can be, for example, ester, ether, phosphoester, amide,peptide, imide, carbon-sulfur bonds, carbon-phosphorus bonds, and thelike. The term “bound” is broader than and includes terms such as“coupled,” “fused” and “attached”.

Core particle: As used herein, the term “core particle” refers to arigid structure with an inherent repetitive organization that provides afoundation for attachment of the first attachment site. A core particleas used herein may be the product of a synthetic process or the productof a biological process.

Coat protein(s): As used herein, the term “coat protein(s)” refers tothe protein(s) of a bacteriophage or a RNA-phage capable of beingincorporated within the capsid assembly of the bacteriophage or theRNA-phage. However, when referring to the specific gene product of thecoat protein gene of RNA-phages the term “CP” is used. For example, thespecific gene product of the coat protein gene of RNA-phage Qβ isreferred to as “Qβ CP”, whereas the “coat proteins” of bacteriophage Qbcomprise the “Qβ CP” as well as the accessory A1 protein. The capsid ofBacteriophage Qβ is composed mainly of the Qβ CP, with a minor contentof the A1 protein. Likewise, the VLP Qβ coat protein contains mainly QβCP, with a minor content of A1 protein.

Conjugate: As used herein, the noun “conjugate” refers to the product ofconjugation between one or more of (a) a core particle such as VLP, andone or more of (b) an organic molecule, hapten, antigen or antigenicdeterminant as described elsewhere herein, wherein the elements (a) and(b) are bound to each other.

Composition: As used herein, the term “composition” refers to a productof mixing or combining various elements or ingredients.

Disease, disorder, condition: As used herein, the terms “disease” or“disorder” refer to any adverse condition of an individual includingtumors, cancer, allergies, addiction, autoimmunity, poisoning orimpairment of optimal mental or bodily function. “Conditions” as usedherein includes diseases and disorders but also refers to physiologicstates. For example, fertility is a physiologic state but not a diseaseor disorder. Compositions of the invention suitable for preventingpregnancy by decreasing fertility would therefore be described as atreatment of a condition (fertility), but not a treatment of a disorderor disease. Other conditions are understood by those of ordinary skillin the art.

Effective Amount: As used herein, the term “effective amount” refers toan amount necessary or sufficient to realize a desired biologic effect.An effective amount of the composition would be the amount that achievesthis selected result, and such an amount could be determined as a matterof routine by a person skilled in the art. For example, an effectiveamount for treating an immune system deficiency could be that amountnecessary to cause activation of the immune system, resulting in thedevelopment of an antigen specific immune response upon exposure toantigen. The term is also synonymous with “sufficient amount.”

The effective amount for any particular application can vary dependingon such factors as the disease or condition being treated, theparticular composition being administered, the size of the subject,and/or the severity of the disease or condition. One of ordinary skillin the art can empirically determine the effective amount of aparticular composition of the present invention without necessitatingundue experimentation.

Epitope: As used herein, the term “epitope” refers to basic element orsmallest unit of recognition by an individual antibody or T-cellreceptor, and thus the particular domain, region or molecular structureto which said antibody or T-cell receptor binds. An antigen may consistof numerous epitopes while a hapten, typically, may possess fewepitopes.

Fusion: As used herein, the term “fusion” refers to the combination ofamino acid sequences of different origin in one polypeptide chain byin-frame combination of their coding nucleotide sequences. The term“fusion” explicitly encompasses internal fusions, i.e., insertion ofsequences of different origin within a polypeptide chain, in addition tofusion to one of its termini.

Hapten: As used herein, the term “hapten” refers to a low-molecularweight organic compound that is not capable of eliciting an immuneresponse by itself but will elicit an immune response once attached to acarrier molecule. Exemplary haptens used in conjugates, compositions andmethods of the invention include drugs, hormones and toxins, but are notlimited to these specific haptens.

Heterologous sequence: As used herein, the term “heterologous sequence”refers to a second sequence of nucleic acid or protein that is notnormally found with said nucleic acid or protein and is, usually,artificially added to the sequence in order to confer particularproperties. In one example, heterologous amino acids may be added torecombinant capsid proteins for the purposes of purification of theprotein, or to serve as a first attachment site.

Isolated: As used herein, when the term “isolated” is used in referenceto a molecule, the term means that the molecule has been removed fromits native environment. For example, a polynucleotide or a polypeptidenaturally present in a living animal is not “isolated,” but the samepolynucleotide or polypeptide separated from the coexisting materials ofits natural state is “isolated.” Further, recombinant DNA moleculescontained in a vector are considered isolated for the purposes of thepresent invention. Isolated RNA molecules include in vivo or in vitroRNA replication products of DNA and RNA molecules. Isolated nucleic acidmolecules further include synthetically produced molecules.Additionally, vector molecules contained in recombinant host cells arealso isolated. Thus, not all “isolated” molecules need be “purified.”

Immune response: As used herein, the term “immune response” refers to ahumoral immune response and/or cellular immune response leading to theactivation or proliferation of B- and/or T-lymphocytes and/or andantigen presenting cells. In some instances, however, the immuneresponses may be of low intensity and become detectable only when usingat least one substance in accordance with the invention. “Immunogenic”refers to an agent used to stimulate the immune system of a livingorganism, so that one or more functions of the immune system areincreased and directed towards the immunogenic agent. An “immunogenicpolypeptide” is a polypeptide that elicits a cellular and/or humoralimmune response, whether alone or linked to a carrier in the presence orabsence of an adjuvant. Preferably, antigen presenting cell may beactivated.

A substance which “enhances” an immune response refers to a substance inwhich an immune response is observed that is greater or intensified ordeviated in any way with the addition of the substance when compared tothe same immune response measured without the addition of the substance.For example, the lytic activity of cytotoxic T cells can be measured,e.g. using a ⁵¹Cr release assay, in samples obtained with and withoutthe use of the substance during immunization. The amount of thesubstance at which the CTL lytic activity is enhanced as compared to theCTL lytic activity without the substance is said to be an amountsufficient to enhance the immune response of the animal to the antigen.In a preferred embodiment, the immune response in enhanced by a factorof at least about 2, more preferably by a factor of about 3 or more. Theamount or type of cytokines secreted may also be altered. Alternatively,the amount of antibodies induced or their subclasses may be altered.

Immunization: As used herein, the terms “immunize” or “immunization” orrelated terms refer to conferring the ability to mount a substantialimmune response (comprising antibodies and/or cellular immunity such aseffector CTL) against a target antigen or epitope. These terms do notrequire that complete immunity be created, but rather that an immuneresponse be produced which is substantially greater than baseline. Forexample, a mammal may be considered to be immunized against a targetantigen if the cellular and/or humoral immune response to the targetantigen occurs following the application of methods of the invention.

Immunotherapeutic: As used herein, the term “immunotherapeutic” refersto a composition for the treatment of diseases, disorders or conditions.More specifically, the term is used to refer to a method of treatmentwherein a beneficial immune response is generated by vaccination or bytransfer of immune molecules.

Immunologically effective amount: As used herein, the term“immunologically effective amount” refers to an amount of a compositionsufficient to induce an immune response in an individual when introducedinto that individual. In the context of active immunization, the term issynonymous with “immunogenically effective amount.” The amount of acomposition necessary to be immunologically effective varies accordingmany factors including to the composition, the presence of othercomponents in the composition (e.g. adjuvants), the antigen, the routeof immunization, the individual, the prior immune or physiologic stateetc.

Individual: As used herein, the term “individual” refers tomulticellular organisms and includes both plants and animals. Preferredmulticellular organisms are animals, more preferred are vertebrates,even more preferred are mammals, and most preferred are humans.

Low or undetectable: As used herein, the phrase “low or undetectable,”when used in reference to gene expression level, refers to a level ofexpression which is either significantly lower than that seen when thegene is maximally induced (e.g., at least five fold lower) or is notreadily detectable by the methods used in the following examplessection.

Lectin: As used herein, proteins obtained particularly from the seeds ofleguminous plants, but also from many other plant and animal sources,that have binding sites for specific mono- or oligosaccharides. Examplesinclude concanavalin A and wheat-germ agglutinin, which are widely usedas analytical and preparative agents in the study of glycoprotein.

Natural origin: As used herein, the term “natural origin” means that thewhole or parts thereof are not synthetic and exist or are produced innature.

Non-natural: As used herein, the term generally means not from nature,more specifically, the term means from the hand of man.

Non-natural origin: As used herein, the term “non-natural origin”generally means synthetic or not from nature; more specifically, theterm means from the hand of man.

Non-natural molecular scaffold: As used herein, the phrase “non-naturalmolecular scaffold” refers to any product made by the hand of man thatserves to provide a rigid and repetitive array of first attachmentsites. Ideally but not necessarily, these first attachment sites are ina geometric order. The non-natural molecular scaffold may be organic ornon-organic and may be synthesized chemically or through a biologicalprocess, in part or in whole. The non-natural molecular scaffold iscomprised of: (a) a core particle, either of natural or non-naturalorigin; and (b) at least one first attachment site that is connected toa core particle by at least one covalent bond. In a particularembodiment, the non-natural molecular scaffold may be a virus,virus-like particle, a bacterial pilus, a virus capsid particle, aphage, a recombinant form thereof, or synthetic particle.

Nicotine hapten: The term “nicotine hapten” as used in the presentinvention refers to nicotine, either in its enantiomerically pure (S)-or (R)-form or a mixture thereof, which could be derivatized in suchmanner as to contain at least one second attachment site which, then, iscapable of associating with the first attachment site of the carriereither directly, or via a cross-linker. Preferably, the nicotine haptenis derivatized in such manner as to contain only one second attachmentsite. This derivatization further increases the order and repetitivenessof the nicotine hapten-carrier conjugate and ensures a directed andcontrolled coupling of the nicotine hapten to the carrier.

Ordered and repetitive antigen or antigenic determinant array: As usedherein, the term “ordered and repetitive antigen or antigenicdeterminant array” generally refers to a repeating pattern of antigen orantigenic determinant, characterized by a uniform spacial arrangement ofthe antigens or antigenic determinants with respect to the non-naturalmolecular scaffold. In one embodiment of the invention, the repeatingpattern may be a geometric pattern. Typical and preferred examples ofsuitable ordered and repetitive antigen or antigenic determinant arraysare those which possess strictly repetitive paracrystalline orders ofantigens or antigenic determinants, preferably with spacings of 0.5 to30 nanometers, more preferably with spacings of 5 to 15 nanometers.

Passive immunization: as used herein, the term “passive immunization”refers to conferral of immunity by the administration, by any route, ofexogenously produced immune molecules (e.g. antibodies) or cells (e.g.T-cells) into an animal. Passive immunization differs from “active”immunization, where immunity is obtained by introduction of animmunogen, vaccine, antigen or hapten-carrier conjugate into anindividual to elicit an immune response.

Pili: As used herein, the term “pili” (singular being “pilus”) refers toextracellular structures of bacterial cells composed of protein monomers(e.g., pilin monomers) which are organized into ordered and repetitivepatterns. Further, pili are structures which are involved in processessuch as the attachment of bacterial cells to host cell surfacereceptors, inter-cellular genetic exchanges, and cell-cell recognition.Examples of pili include Type-1 pili, P-pili, F1C pili, S-pili, and987P-pili. Additional examples of pili are set out elsewhere herein.

Pilus-like structure: As used herein, the phrase “pilus-like structure”refers to structures having characteristics similar to that of pili andcomposed of protein monomers. One example of a “pilus-like structure” isa structure formed by a bacterial cell which expresses modified pilinproteins that do not form ordered and repetitive arrays that areessentially identical to those of natural pili.

Polypeptide: As used herein, the term “polypeptide” refers to a moleculecomposed of monomers (amino acids) linearly linked by amide bonds (alsoknown as peptide bonds). It indicates a molecular chain of amino acidsand does not refer to a specific length of the product. Thus, peptides,dipeptides, tripeptides, oligopeptides and proteins are included withinthe definition of polypeptide. This term is also intended to refer topost-expression modifications of the polypeptide, for example,glycosolations, acetylations, phosphorylations, and the like. Arecombinant or derived polypeptide is not necessarily translated from adesignated nucleic acid sequence. It may also be generated in anymanner, including chemical synthesis.

Protein: As used herein, the term protein refers to a polypeptidegenerally of a size of above about 5 or more, 10 or more 20 or more, 25or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more,1000 or more, 2000 or more amino acids. Proteins generally have adefined three dimensional structure although they do not necessarilyneed to, and are often referred to as folded, as opposed to peptides andpolypeptides which often do not possess a defined three-dimensionalstructure, but rather can adopt a large number of differentconformations, and are referred to as unfolded. Peptides may, however,adopt three dimensional structures. The defined three-dimensionalstructures of proteins is especially important for the associationbetween the core particle and the antigen, mediated by the secondattachment site, and in particular by way of chemical cross-linkingbetween the first and second attachment site using a chemicalcross-linker. The amino acid linker is also intimately related to thestructural properties of proteins in some aspects of the invention.

Purified: As used herein, when the term “purified” is used in referenceto a molecule, it means that the concentration of the molecule beingpurified has been increased relative to molecules associated with it inits natural environment, or environment in which it was produced, foundor synthesized. Naturally associated molecules include proteins, nucleicacids, lipids and sugars but generally do not include water, buffers,and reagents added to maintain the integrity or facilitate thepurification of the molecule being purified. For example, even if mRNAis diluted with an aqueous solvent during oligo dT columnchromatography, mRNA molecules are purified by this chromatography ifnaturally associated nucleic acids and other biological molecules do notbind to the column and are separated from the subject mRNA molecules.According to this definition, a substance may be 5% or more, 10% ormore, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more,70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% ormore, or 100% pure when considered relative to its contaminants.

Receptor: As used herein, the term “receptor” refers to proteins orglycoproteins or fragments thereof capable of interacting with anothermolecule, called the ligand. The ligand may belong to any class ofbiochemical or chemical compounds. The receptor need not necessarily bea membrane-bound protein. Soluble protein, like e.g., maltose bindingprotein or retinol binding protein are receptors as well.

Residue: As used herein, the term “residue” is meant to mean a specificamino acid in a polypeptide backbone or side chain.

Recombinant host cell: As used herein, the term “recombinant host cell”refers to a host cell into which one ore more nucleic acid molecules ofthe invention have been introduced.

Recombinant virus: As used herein, the phrase “recombinant virus” refersto a virus that is genetically modified by the hand of man. The phrasecovers any virus known in the art. More specifically, the phrase refersto a an alphavirus genetically modified by the hand of man, and mostspecifically, the phrase refers to a Sinbis virus genetically modifiedby the hand of man.

RNA-phage, RNA-bacteriophage: As used herein, the term“RNA-bacteriophage,” or its abbreviated form “RNA-phage” refers to RNAviruses infecting bacteria, preferably to single-stranded positive-senseRNA viruses infecting bacteria.

Self antigen: As used herein, the tem “self antigen” refers to proteinsencoded by the host's DNA and products generated by proteins or RNAencoded by the host's DNA are defined as self. In addition, proteinsthat result from a combination of two or several self-molecules or thatrepresent a fraction of a self-molecule and proteins that have a highhomology two self-molecules as defined above (>95%, preferably >97%,more preferably >99%) may also be considered self.

Vaccine: As used herein, the term “vaccine” refers to a formulationwhich contains the composition of the present invention and which is ina form that is capable of being administered to an animal. Typically,the vaccine comprises a conventional saline or buffered aqueous solutionmedium in which the composition of the present invention is suspended ordissolved. In this form, the composition of the present invention can beused conveniently to prevent, ameliorate, or otherwise treat acondition. Upon introduction into a host, the vaccine is able to provokean immune response including, but not limited to, the production ofantibodies and/or cytokines and/or the activation of cytotoxic T cells,antigen presenting cells, helper T cells, dendritic cells and/or othercellular responses. Optionally, the vaccine of the present inventionadditionally includes an adjuvant which can be present in either a minoror major proportion relative to the compound of the present invention.

Vector: As used herein, the term “vector” refers to an agent (e.g., aplasmid or virus) used to transmit genetic material to a host cell. Avector may be composed of either DNA or RNA.

Virus-like particle: As used herein, the term “virus-like particle”refers to a structure resembling a virus particle. Moreover, avirus-like particle in accordance with the invention is non replicativeand noninfectious since it lacks all or part of the viral genome, inparticular the replicative and infectious components of the viralgenome. A virus-like particle in accordance with the invention maycontain nucleic acid distinct from their genome.

Virus-like particle of a bacteriophage: As used herein, the term“virus-like particle of a bacteriophage” refers to a virus-like particleresembling the structure of a bacteriophage, being non replicative andnoninfectious, and lacking at least the gene or genes encoding for thereplication machinery of the bacteriophage, and typically also lackingthe gene or genes encoding the protein or proteins responsible for viralattachment to or entry into the host. A virus-like particle in accodancewith the invention lacks all or part of the viral genome, in particularthe replicative and infectious components of the viral genome. Avirus-like particle in accordance with the invention may contain nucleicacid distinct from their genome. A typical and preferred embodiment of avirus-like particle in accordance with the present invention is a viralcapsid such as the viral capsid of the corresponding virus,bacteriophage, or RNA-phage. The terms “viral capsid” or “capsid”, asinterchangeably used herein, refer to a macromolecular assembly composedof viral protein subunits. Typically and preferably, the viral proteinsubunits assemble into a viral capsid and capsid, respectively, having astructure with an inherent repetitive organization, wherein saidstructure is, typically, spherical or tubular. For example, the capsidsof RNA-phages or HBcAg's have a spherical form of icosahedral symmetry.The term “capsid-like structure” as used herein, refers to amacromolecular assembly composed of viral protein subunits ressemblingthe capsid morphology in the above defined sense but deviating from thetypical symmetrical assembly while maintaining a sufficient degree oforder and repetitiveness.

Virus-like particle of a bacteriophage: As used herein, the term“virus-like particle of a bacteriophage” refers to a virus-like particleresembling the structure of a bacteriophage, being non replicative andnoninfectious, and lacking at least the gene or genes encoding for thereplication machinery of the bacteriophage, and typically also lackingthe gene or genes encoding the protein or proteins responsible for viralattachment to or entry into the host. This definition should, however,also encompass virus-like particles of bacteriophages, in which theaforementioned gene or genes are still present but inactive, and,therefore, also leading to non-replicative and noninfectious virus-likeparticles of a bacteriophage.

VLP of RNA phage coat protein: The capsid structure formed from theself-assembly of 180 subunits of RNA phage coat protein and optionallycontaining host RNA is referred to as a “VLP of RNA phage coat protein”.A specific example is the VLP of Qβ coat protein. In this particularcase, the VLP of Qβ coat protein may either be assembled exclusivelyfrom Qβ CP subunits (generated by expression of a Qβ CP gene containing,for example, a TAA stop codon precluding any expression of the longer A1protein through suppression, see Kozlovska, T. M., et al., Intervirology39: 9-15 (1996)), or additionally contain A1 protein subunits in thecapsid assembly.

Virus particle: The term “virus particle” as used herein refers to themorphological form of a virus. In some virus types it comprises a genomesurrounded by a protein capsid; others have additional structures (e.g.,envelopes, tails, etc.).

One, A, or An: When the terms “one,” “a” or “an” are used in thisdisclosure, they mean “at least one” or “one or more” unless otherwiseindicated.

As used herein when referring to any numerical value, the term “about”means a value of ±10% of the stated value (e.g., “about 50° C.”encompasses a range of temperatures from 45° C. to 55° C., inclusive;similarly, “about 100 mM” encompasses a range of concentrations from 90mM to 110 mM inclusive).

Overview

In one aspect, the invention provides conjugates of one or more haptenswith a carrier in an ordered and repetitive hapten-carrier conjugate,and methods of making such conjugates. The invention also providescompositions comprising at least one such conjugate of the invention andat least one other component, suitably at least one excipient or carrierand particularly at least one pharmaceutically acceptable excipient orcarrier. Haptens suitably used in the conjugates and compositions of theinvention include but are not limited to hormones, toxins and drugs,especially drugs of addiction, such as nicotine. The conjugates andcompositions of the invention are useful for inducing immune responsesagainst haptens. Such an immune response can be utilized to generateantibodies, useful for therapeutic, prophylactic and diagnosticpurposes. Immune response can be useful to prevent or treat addiction todrugs of abuse and the resultant diseases associated with drugaddiction.

The conjugates of the present invention comprise highly ordered andrepetitive arrays of haptens. Conjugate arrays according to this aspectof the invention comprise (a) a core particle, comprising a firstattachment site and (b) a hapten comprising a second attachment site,wherein the elements (a) and (b) are linked through the first and secondattachment sites to form said ordered and repetitive hapten arrays.

Core particles suitably used in the conjugates and compositions of theinvention may be natural or non-natural. Natural core particles of thepresent invention include virus particles, virus-like particles, andpili. The proteins of these natural core particles may be natural orrecombinant. The first attachment sites on the core particle may occurnaturally or may be introduced via chemical or recombinant means.Haptens of the present invention are those suitable for inducing immuneresponses against a variety of molecules, including but not limited totoxins, hormones and drugs, particularly drugs of abuse and oraddiction. The second attachment site on the hapten may naturally occuror be introduced. The interaction between first and second sites may bedirect, or may involve at least one other molecule, e.g. a linker.Linkers include cross-linking molecules.

The conjugates and compositions of the invention are suprisinglyeffective in inducing immune responses, particularly antibodies, againsthaptens. Thus, they are useful in compositions suitable for immunizationof animals for therapeutic or prophylaxis against diseases, disorders orconditions associated with various drugs, hormones or toxins. Antibodiesproduced by immunization with the conjugates and compositions of theinvention are also useful for therapeutic and prophylactic purposes.

In other embodiments, the invention provides methods of treatment andprevention of a disease utilizing the conjugates and compositions of theinvention. In another embodiment, the invention provides kits suitablefor diagnosis and screening.

Compositions of Ordered and Repetitive Antigen or Antigenic DeterminantArrays and Methods to Make the Same

The present invention provides conjugates, and compositions ofconjugates, comprising an ordered and repetitive hapten array.Furthermore, the invention conveniently enables the practitioner toconstruct ordered and repetitive hapten arrays for various purposes, andpreferably the induction of an immune response against organicmolecules.

Conjugates of the invention essentially comprise, or alternativelyconsist of, two elements: (1) a non-natural molecular scaffold; and (2)a hapten with at least one second attachment site capable of associationthrough at least one bond to said first attachment site.

The non-natural molecular scaffold comprises, or alternatively consistsof: (a) a core particle selected from the group consisting of (1) a coreparticle of non-natural origin and (2) a core particle of naturalorigin; and (b) at least one first attachment site connected to saidcore particle by at least one covalent bond. Core particles used in theconjugates, compositions and methods of the invention include inorganicmolecules, virus particles, virus-like particles, and bacterial pili.The haptens used in the conjugates, compositions and methods of theinvention has at least one second attachment site which is selected fromthe group consisting of (a) an attachment site not naturally occurringwith said hapten; and (b) an attachment site naturally occurring withsaid antigen or antigenic determinant.

The invention provides for an ordered and repetitive hapten arraythrough an association of the second attachment site to the firstattachment site by way of at least one bond. Thus, the hapten and thenon-natural molecular scaffold are brought together through thisassociation of the first and the second attachment site to form anordered and repetitive antigen array.

The practioner may specifically design the hapten and the secondattachment site such that the arrangement of all the haptens bound tothe non-natural molecular scaffold or, in certain embodiments, the coreparticle will be uniform. For example, one may place a single secondattachment site on the hapten, thereby ensuring through design that allhaptens that are attached to the non-natural molecular scaffold arepositioned in a uniform way. Thus, the invention provides a convenientmeans of placing any hapten onto a non-natural molecular scaffold in adefined order and in a manner which forms a repetitive pattern.

As will be clear to those of ordinary skill in the art, certainembodiments of the invention involve the use of recombinant nucleic acidtechnologies such as cloning, polymerase chain reaction, thepurification of DNA and RNA, the expression of recombinant proteins inprokaryotic and eukaryotic cells, etc. Such methodologies are well knownto those skilled in the art and may be conveniently found in publishedlaboratory methods manuals (e.g., Sambrook, J. et al., eds., MOLECULARCLONING, A LABORATORY MANUAL, 2nd. edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel, F. et al.,eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John H. Wiley & Sons, Inc.(1997)). Fundamental laboratory techniques for working with tissueculture cell lines (Celis, J., ed., CELL BIOLOGY, Academic Press, 2^(nd)edition, (1998)) and antibody-based technologies (Harlow, E. and Lane,D., “Antibodies: A Laboratory Manual,” Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1988); Deutscher, M. P., “Guide to ProteinPurification,” Meth. Enzymol. 128, Academic Press San Diego (1990);Scopes, R. K., “Protein Purification Principles and Practice,” 3^(rd)ed., Springer-Verlag, New York (1994)) are also adequately described inthe literature, all of which are incorporated herein by reference.

Furthermore, technologies for coupling organic molecules to amino acidsand means for making derivatives of haptens containing appropriatesecond attachment sites such as are neccessary for the practice of theinvention are well known to those of skill in the art. Suchmethodologies may be found in chemical text books and publications,examples of which are included below and are incorportated by reference;U.S. Pat. No. 5,876,727; WO 99/61054; Isomura, S. et al. J. Org. Chem.66:4115-4121 (2001); Matsushita, H. et al. Biochem. Biophys. Res. Comm.57:1006-1010. (1974); Langone, J. L. and Van Vunakis, H., MethodsEnzymol. 84:628-640 (1982); Wong, Chemistry of Protein Conjugation andCross-Linking. CRC Press, Inc., Boca Raton, Fla. (1991.)

Core Particles and Non-Natural Molecular Scaffolds

In one embodiment, the present invention provides methods for theformation of an ordered and repetitive array of haptens. By theinvention, this occurs by the association of a core particle to which isattached one or more haptens via first and second attachment sites.

Thus, one element in certain conjugates and compositions of theinvention is a non-natural molecular scaffold comprising, oralternatively consisting of, a core particle and a first attachmentsite. More specifically, the non-natural molecular scaffold comprises,or alternatively consists of, (a) a core particle of natural ornon-natural origin and (b) at least one first attachment site connectedto the core particle by at least one covalent bond.

Core particles. In one embodiment of the present invention, a coreparticle is a synthetic polymer, a lipid micelle or a metal. Such coreparticles are known in the art, providing a basis from which to buildthe non-natural molecular scaffold of the invention. By way of example,synthetic polymer or metal core particles are disclosed in U.S. Pat. No.5,770,380, and U.S. Pat. No. 5,334,394, which are incorporated byreference herein in their entirities. Suitable metals include, but arenot limited to, chromium, rubidium, iron, zinc, selenium, nickel, gold,silver, platinum. Suitable ceramic materials include, but are notlimited to, silicon dioxide, titanium dioxide, aluminum oxide, rutheniumoxide and tin oxide. The core particles of this embodiment may be madefrom organic materials including, but not limited to, carbon andsuitable polymers, including polystyrene, nylon and nitrocellulose. Fornanocrystalline particles, particles made from tin oxide, titaniumdioxide or carbon (diamond) are useful. Lipid micelles for use in thepresent invention are prepared by any means known in the art, forexample, Baiselle and Millar (Biophys. Chem. 4:355-361 (1975)) or Cortiet al. (Chem. Phys. Lipids 38:197-214 (1981)) or Lopez et al. (FEBSLett. 426:314-318 (1998)) or Topchieva and Karezin (J. Colloid InterfaceSci. 213:29-35 (1999)) or Morein et al., (Nature 308:457-460 (1984)),which are incorporated herein by reference in their entirities.

In one embodiment of the invention the core particle is produced througha biological process, which may be natural or non-natural. For example,viruses and bacterial pili or pilus-like structures are formed fromproteins which are organized into ordered and repetitive structures.Therefore, the present invention comprises conjugates, compositions andmethods comprising useful core particles which include, but are notlimited to a virus, virus-like particle, a bacterial pilus, a phage, aviral capsid particle or fragments thereof. In certain such embodiments,the proteins may be recombinant.

In certain embodiments, the core particle of the non-natural molecularscaffold comprises a virus, a bacterial pilus, a structure formed frombacterial pilin, a bacteriophage, a virus-like particle, a viral capsidparticle or a recombinant form thereof. Any virus known in the arthaving an ordered and repetitive coat and/or core protein structure maybe selected for use as in the methods, conjugates and compositions ofthe invention as a non-natural molecular scaffold. Examples of suitableviruses include, but are not limited to, sindbis and other alphaviruses,rhabdoviruses (e.g. vesicular stomatitis virus), picornaviruses (e.g.,human rhino virus, Aichi virus), togaviruses (e.g., rubella virus),orthomyxoviruses (e.g., Thogoto virus, Batken virus, fowl plague virus),polyomaviruses (e.g., polyomavirus BK, polyomavirus JC, avianpolyomavirus BFDV), parvoviruses, rotaviruses, bacteriophage Qβ,bacteriophage R17, bacteriophage M11, bacteriophage MX1, bacteriophageNL95, bacteriophage fr, bacteriophage GA, bacteriophage SP,bacteriophage MS2, bacteriophage f2, bacteriophage PP7, bacteriophageAP205, Norwalk virus, foot and mouth disease virus, a retrovirus,Hepatitis B virus, Tobacco mosaic virus, Flock House Virus, and humanPapilomavirus (for example, see Table 1 in Bachman, M. F. andZinkernagel, R. M., Immunol. Today 17:553-558 (1996)). In more specificexemplary embodiments of the present invention the core particle maycomprise, or alternatively consist of, recombinant proteins ofRotavirus, recombinant proteins of Norwalk virus, recombinant proteinsof Alphavirus, recombinant proteins which form bacterial pili orpilus-like structures, recombinant proteins of Foot and Mouth Diseasevirus, recombinant proteins of Retrovirus, recombinant proteins ofHepatitis B virus (e.g., a HBcAg), recombinant proteins of Tobaccomosaic virus, recombinant proteins of Flock House Virus, and recombinantproteins of human Papillomavirus.

The core particle used in conjugates, compositions and methods of theinvention may further comprise, or alternatively consist of, one or morefragments of such proteins, as well as variants of such proteins whichretain the ability to associate with each other to form ordered andrepetitive antigen or antigenic determinant arrays. For example, asexplained in WO 02/056905 core particles may be formed from variantforms of the human HBcAg which differ markedly from the wild-typeparticle in amino acid sequence identity and similarity, and in sequencelength. For example, amino acid sequence of the HBcAg of Hepatitis Bviruses which infect snow geese and ducks differs sufficiently from thatof HBcAg of viruses infected mammals that alignment of the proteins isdifficult. However, both viruses retain the ability to form corestructures suitable for the formation of ordered repetitive haptenarrays. Similarly, HBcAg may retain the ability to form multimericparticles, typical of a virus, after removal of N-terminal leadersequences, further deletions, substitutions, or additions to thesequence. Methods which can be used to determine whether proteins formsuch structures comprise gel filtration, agarose gel electrophoresis,sucrose gradient centrifugation and electron microscopy (e.g., Koschel,M. et al., J. Virol 73: 2153-2160 (1999)).

First Attachment Sites. Whether natural or non-natural, the coreparticle used in the conjugates, compositions and methods of the presentinvention will generally possess a component comprising a firstattachment site that is attached to the natural or non-natural coreparticle by at least one covalent bond. The element comprising the firstattachment site is bound to a core particle in a non-random fashion thatprovides a nucleation site for creating an ordered and repetitiveantigen array. Ideally, but not necessarily, this element is associatedwith the core particle in a geometric order. The first attachment sitemay be a natural part of the core particle, such as a surface exposedamino acid residue suitable for coupling to the second attachment site.For example, lysine and cysteine may form non-peptide bonds via reactivegroups on the amino acid. Alternatively, an element containing the firstattachment site may be introduced into the core particle via chemicalcoupling or through the design of recombinant molecules. The firstattachment site may be, or be found on, any element comprising bound toa core particle by at least one covalent bond.

Elements comprising, or alternatively consisting of, the firstattachment site may be proteins, a polypeptide, a peptide, an amino acid(i.e., a residue of a protein, a polypeptide or peptide), a sugar, apolynucleotide, a natural or synthetic polymer, a secondary metaboliteor compound (biotin, fluorescein, retinol, digoxigenin, metal ions,phenylmethylsulfonylfluoride), or a combination thereof, or a chemicallyreactive group thereof. In a more specific embodiment, the firstattachment site comprising an antigen, an antibody or antibody fragment,biotin, avidin, strepavidin, a receptor, a receptor ligand, a ligand, aligand-binding protein, an interacting leucine zipper polypeptide, anamino group, a chemical group reactive to an amino group; a carboxylgroup, chemical group reactive to a carboxyl group, a sulfhydryl group,a chemical group reactive to a sulfhydryl group, or a combinationthereof.

In one embodiment, the invention utilizes genetic engineering of a virusto create a fusion between an ordered and repetitive viral envelopeprotein the element comprising the first attachment site whichcomprising a heterologous protein, peptide, antigenic determinant or areactive amino acid residue of choice. Other genetic manipulations knownto those in the art may be included in the construction of thenon-natural molecular scaffold; for example, it may be desirable torestrict the replication ability of the recombinant virus throughgenetic mutation. The viral protein selected for fusion to the proteincontaining the first attachment site protein should have an organizedand repetitive structure. Such an organized and repetitive structureinclude paracrystalline organizations with a spacing of 0.5-30,preferably 5-15 nm, on the surface of the virus. The creation of thistype of fusion protein will result in multiple, ordered and repetitivefirst attachment sites on the surface of the virus. Thus, the orderedand repetitive organization of the first attachment sites resultingtherefrom will reflect the normal organization of the native viralprotein.

As will be understood by those of ordinary skill in the art, the firstattachment site may be or be a part of any suitable protein,polypeptide, sugar, polynucleotide, peptide (amino acid), natural orsynthetic polymer, a secondary metabolite or combination thereof thatmay serve to specifically attach the antigen or antigenic determinant ofchoice to the non-natural molecular scaffold. In one embodiment, theattachment site is a protein or peptide that may be selected from thoseknown in the art. For example, the first attachment site may be aligand, a receptor, a lectin, avidin, streptavidin, biotin, an epitopesuch as an HA or T7 tag, Myc, Max, immunoglobulin domains and any otheramino acid sequence known in the art that would be useful as a firstattachment site.

It will be further understood by those of ordinary skill in the art thatwith another embodiment of the invention, the first attachment site maybe created secondarily to the creation of an element carrying the firstattachment site (i.e., protein or polypeptide) utilized in constructingthe in-frame fusion to the capsid protein. For example, a protein may beutilized for fusion to the envelope protein with an amino acid sequenceknown to be glycosylated in a specific fashion, and the sugar moietyadded as a result may then serve at the first attachment site of theviral scaffold by way of binding to a lectin serving as the secondaryattachment site of an antigen. Alternatively, a sequence may bebiotinylated in vivo and the biotin moiety may serve as the firstattachment site of the invention, or the sequence may be subjected tochemical modification of distinct amino acid residues in vitro, themodification serving as the first attachment site.

In one specific embodiment of the invention, the first attachment siteis the JUN-FOS leucine zipper protein domain that is fused in frame tothe Hepatitis B capsid (core) protein (HBcAg). However, it will be clearto those of ordinary skill in the art that other viral capsid proteinsmay be utilized in the fusion protein construct for locating the firstattachment site in the non-natural molecular scaffold of the invention.For example, in other embodiments of the invention, the first attachmentsite is selected to be a lysine or cysteine residue that is fused inframe to the HBcAg. However, it will be clear to all individuals in theart that other viral capsid or virus-like particles may be utilized inthe fusion protein construct for locating the first attachment in thenon-natural molecular scaffold of the invention.

Viral particles. In one embodiment of the invention, the core particleis a recombinant alphavirus, and more specifically, a recombinantSindbis virus. Several members of the alphavirus family, Sindbis (Xiong,C. et al., Science 243:1188-1191 (1989); Schlesinger, S., TrendsBiotechnol. 11:18-22 (1993)), Semliki Forest Virus (SFV) (Liljeström, P.& Garoff, H., Bio/Technology 9:1356-1361 (1991)) and others (Davis, N.L. et al., Virology 171:189-204 (1989)), have received considerableattention for use as virus-based expression vectors for a variety ofdifferent proteins (Lundstrom, K., Curr. Opin. Biotechnol. 8:578-582(1997); Liljeström, P., Curr. Opin. Biotechnol. 5:495-500 (1994)) and ascandidates for vaccine development. The use of alphaviruses for theexpression of heterologous proteins and the development of vaccines hasbeen disclosed (see U.S. Pat. Nos. 5,766,602; 5,792,462; 5,739,026;5,789,245; and 5,814,482) the disclosures all of which are incorporatedby reference in their entirities. The construction of the alphaviralscaffold of the invention may be done by means generally known in theart of recombinant DNA technology, as described by the aforementionedarticles, which are incorporated herein by reference. A variety ofdifferent recombinant host cells can be utilized to produce aviral-based core particle for antigen or antigenic determinantattachment.

Packaged RNA sequences can also be used to infect host cells. Thesepackaged RNA sequences can be introduced to host cells by adding them tothe culture medium. For example, the preparation of non-infectivealpahviral particles is described in a number of sources, including“Sindbis Expression System”, Version C (Invitrogen Corporation, CarlsbadCalif.; Catalog No. K750-1).

When mammalian cells are used as recombinant host cells for theproduction of viral-based core particles, these cells will generally begrown in tissue culture. Methods for growing cells in culture are wellknown in the art (see, e.g., Celis, J., ed., CELL BIOLOGY, AcademicPress, 2^(nd) edition, (1998); Sambrook, J. et al., eds., MOLECULARCLONING, A LABORATORY MANUAL, 2nd. edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel, F. et al.,eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John H. Wiley & Sons, Inc.(1997); Freshney, R., CULTURE OF ANIMAL CELLS, Alan R. Liss, Inc.(1983)).

The invention thus includes viral-based core particles which comprise,or alternatively consist of, a virus, virus-like particle, a phage, aviral capsid particle or a recombinant form thereof. Skilled artisanshave the knowledge to produce such core particles and attach firstattachment sites thereto. The production of Hepatitis B virus-likeparticles, in particular those assembled or self-assembled from HBcAg,and measles viral capsid particles as core particles is disclosed inExamples 17 to 22 of WO 00/32227, which is explicitly incorporatedherein by reference. In such embodiments, the JUN leucine zipper proteindomain or FOS leucine zipper protein domain may be used as a firstattachment site for the non-natural molecular scaffold of the invention.One of skill in the art would known methods for constructing Hepatitis Bcore particles carrying an in-frame fused peptide with a reactive lysineresidue and antigens carrying a genetically fused cysteine residue, asfirst and second attachment site, respectively.

In other embodiments, the core particles used in compositions of theinvention are composed of a Hepatitis B capsid (core) protein (HBcAg), afragment of a HBcAg, or other protein or peptide which can formvirus-like particles, which are ordered arrays, which have been modifiedto either eliminate or reduce the number of free cysteine residues. Zhouet al. (J. Virol. 66:5393-5398 (1992)) demonstrated that HBcAgs whichhave been modified to remove the naturally resident cysteine residuesretain the ability to associate and form multimeric structures. Thus,core particles suitable for use in compositions of the invention includethose comprising modified HBcAgs, or fragments thereof, in which one ormore of the naturally resident cysteine residues have been eitherdeleted or substituted with another amino acid residue (e.g., a serineresidue). In a preferred embodiment, the HBcAg has the amino acidsequence as set forth in SEQ ID NO: 1, or a sequence that is at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,more preferably at least about 99% or 100% identical to the sequence ofSEQ ID NO: 1. In one embodiment of the invention, a modified HBcAgcomprising the amino acid sequence shown in SEQ ID NO:1, or subportionthereof, is used to prepare non-natural molecular scaffolds. Inparticular, modified HBcAgs suitable for use in the practice of theinvention include proteins in which one or more of the cysteine residuesat positions corresponding to positions 48, 61, 107 and 185 of a proteinhaving the amino acid sequence shown in SEQ ID NO:1 have been eitherdeleted or substituted with other amino acid residues (e.g., a serineresidue). As one skilled in the art would recognize, cysteine residuesat similar locations in HBcAg variants having amino acids sequenceswhich differ from that shown in SEQ ID NO:1 could also be deleted orsubstituted with other amino acid residues. The modified HBcAg variantscan then be used to prepare vaccine compositions of the invention.

Under certain circumstances (e.g., when a heterobifunctionalcross-linking reagent is used to attach antigens or antigenicdeterminants to the non-natural molecular scaffold), the presence offree cysteine residues in the HBcAg is believed to lead to covalentcoupling of toxic components to core particles, as well as thecross-linking of monomers to form undefined species.

Further, in many instances, these toxic components may not be detectablewith assays performed on compositions of the invention. This is sobecause covalent coupling of toxic components to the non-naturalmolecular scaffold would result in the formation of a population ofdiverse species in which toxic components are linked to differentcysteine residues, or in some cases no cysteine residues, of the HBcAgs.In other words, each free cysteine residue of each HBcAg will not becovalently linked to toxic components. Further, in many instances, noneof the cysteine residues of particular HBcAgs will be linked to toxiccomponents. Thus, the presence of these toxic components may bedifficult to detect because they would be present in a mixed populationof molecules. The administration to an individual of HBcAg speciescontaining toxic components, however, could lead to a potentiallyserious adverse reaction.

It is well known in the art that free cysteine residues can be involvedin a number of chemical side reactions. These side reactions includedisulfide exchanges, reaction with chemical substances or metabolitesthat are, for example, injected or formed in a combination therapy withother substances, or direct oxidation and reaction with nucleotides uponexposure to UV light. Toxic adducts could thus be generated, especiallyconsidering the fact that HBcAgs have a strong tendency to bind nucleicacids. Detection of such toxic products in antigen-capsid conjugateswould be difficult using capsids prepared using HBcAgs containing freecysteines and heterobifunctional cross-linkers, since a distribution ofproducts with a broad range of molecular weight would be generated. Thetoxic adducts would thus be distributed between a multiplicity ofspecies, which individually may each be present at low concentration,but reach toxic levels when together.

In view of the above, one advantage to the use of HBcAgs in vaccinecompositions which have been modified to remove naturally residentcysteine residues is that sites to which toxic species can bind whenantigens or antigenic determinants are attached to the non-naturalmolecular scaffold would be reduced in number or eliminated altogether.Further, a high concentration of cross-linker can be used to producehighly decorated particles without the drawback of generating aplurality of undefined cross-linked species of HBcAg monomers (i.e., adiverse mixture of cross-linked monomeric HbcAgs).

A number of naturally occurring HBcAg variants suitable for use in thepractice of the present invention have been identified. Yuan et al., (J.Virol. 73:10122-10128 (1999)), for example, describe variants in whichthe isoleucine residue at position corresponding to position 97 in SEQID NO:1 is replaced with either a leucine residue or a phenylalanineresidue. The amino acid sequences of a number of HBcAg variants, as wellas several Hepatitis B core antigen precursor variants, are disclosed inGenBank reports AAF121240, AF121239, X85297, X02496, X85305, X85303,AF151735, X85259, X85286, X85260, X85317, X85298, AF043593, M20706,X85295, X80925, X85284, X85275, X72702, X85291, X65258, X85302, M32138,X85293, X85315, U95551, X85256, X85316, X85296, AB033559, X59795, X8529,X85307, X65257, X85311, X85301, X85314, X85287, X85272, X85319,AB010289, X85285, AB010289, AF121242, M90520, P03153, AF110999, andM95589, the disclosures of each of which are incorporated herein byreference. These HBcAg variants differ in amino acid sequence at anumber of positions, including amino acid residues which corresponds tothe amino acid residues located at positions 12, 13, 21, 22, 24, 29, 32,33, 35, 38, 40, 42, 44, 45, 49, 51, 57, 58, 59, 64, 66, 67, 69, 74, 77,80, 81, 87, 92, 93, 97, 98, 100, 103, 105, 106, 109, 113, 116, 121, 126,130, 133, 135, 141, 147, 149, 157, 176, 178, 182 and 183 in SEQ ID NO:1.

Further HBcAg variants suitable for use in the compositions of theinvention, and which may be further modified according to the disclosureof this specification are described in WO 00/198333, WO 00/177158 and WO00/214478, herein included by reference in their entirety.

HBcAgs suitable for use in the present invention may be derived from anyorganism so long as they are able to associate to form an ordered andrepetitive antigen array. Generally processed HBcAgs (i.e., those whichlack leader sequences) will be used in the vaccine compositions of theinvention.The present invention includes vaccine compositions, as wellas methods for using these compositions, which employ the abovedescribed variant HBcAgs for the preparation of non-natural molecularscaffolds. Further included within the scope of the invention areadditional HBcAg variants which are capable of associating to formdimeric or multimeric structures. Thus, the invention further includesvaccine compositions comprising HBcAg polypeptides comprising, oralternatively consisting of, amino acid sequences which are at leastabout 80%, about 85%, about 90%, about 95%, about 97%, or about 99%identical to any of the amino acid sequences shown in the abovesequences, including SEQ ID No: 1, and forms of these proteins whichhave been processed, where appropriate, to remove the N-terminal leadersequence.

Whether the amino acid sequence of a polypeptide has an amino acidsequence that is at least about 80%, about 85%, about 90%, about 95%,about 97%, or about 99% identical to one of the amino acid sequencesshown above, or a subportion thereof, can be determined conventionallyusing known computer programs such the Bestfit program. When usingBestfit or any other sequence alignment program to determine whether aparticular sequence is, for instance, about 95% identical to a referenceamino acid sequence according to the present invention, the parametersare set such that the percentage of identity is calculated over the fulllength of the reference amino acid sequence and that gaps in homology ofup to 5% of the total number of amino acid residues in the referencesequence are allowed. In such a manner, comparisons may be made betweenthe amino acid sequence of HBcAg of SEQ ID NO:1 and other HBcAg. Whencomparing proteins that are relatively similar, reference to an aminoacid residue of a HBcAg variant located at a position which correspondsto a particular position in SEQ ID NO:1, refers to the amino acidresidue which is present at that position in the amino acid sequenceshown in SEQ ID NO:1. The homology between these HBcAg variants is forthe most part high enough among Hepatitis B viruses that infect mammalsso that one skilled in the art would have little difficulty reviewingboth the amino acid sequence shown in SEQ ID NO:1 and that of aparticular HBcAg variant and identifying “corresponding” amino acidresidues. For example, comparisons between the SEQ ID NO:1 and the aminoacid sequence of the an HBcAg derived from a virus which infectwoodchucks, it is readily apparent that a three amino acid residueinsert is present in that sequence between amino acid residues 155 and156 of SEQ ID NO:1.

However, where alignment is difficult, one skilled in the art wouldrecognize the importance of particular amino acids or motifs in asequence. For example, the amino acid sequence of HBcAg from humanviruses differs from duck viruses such that alignment is difficult, yetone skilled in the art would recognize conserved cysteine residues couldbe either substituted with another amino acid residue or deleted priorto their inclusion in vaccine compositions of the invention.

In one embodiment, the cysteine residues at positions 48 and 107 of aprotein having the amino acid sequence shown in SEQ ID NO:1 are deletedor substituted with another amino acid residue but the cysteine atposition 61 is left in place. Further, the modified polypeptide is thenused to prepare vaccine compositions of the invention.

The preparation of preferred Hepatitis B virus-like particles, which canbe used for the present invention, is disclosed, for example, in WO00/32227, and hereby in particular in Examples 17 to 19 and 21 to 24, aswell as in WO 01/85208, and hereby in particular in Examples 17 to 19,21 to 24, 31 and 41, and in WO 02/056905. For the latter application, itis in particular referred to Example 23, 24, 31 and 51. All threedocuments are explicitly incorporated herein by reference.

As set out below in Example 31 of WO 02/056905, the cysteine residues atpositions 48 and 107, which are accessible to solvent, may be removed,for example, by site-directed mutagenesis. Further, the inventors havefound that the Cys-48-Ser, Cys-107-Ser HBcAg double mutant constructedas described in WO 02/056905, (which is incorporated herein by referencein its entirety) can be expressed in E. coli.

As discussed above, the elimination of free cysteine residues reducesthe number of sites where toxic components can bind to the HBcAg, andalso eliminates sites where cross-linking of lysine and cysteineresidues of the same or of neighboring HBcAg molecules can occur. Thecysteine at position 61, which is involved in dimer formation and formsa disulfide bridge with the cysteine at position 61 of another HBcAg,will normally be left intact for stabilization of HBcAg dimers andmultimers of the invention. Cross-linking experiments performed with (1)HBcAgs containing free cysteine residues and (2) HBcAgs whose freecysteine residues have been made unreactive with iodacetamide, indicatethat free cysteine residues of the HBcAg are responsible forcross-linking between HBcAgs through reactions betweenheterobifunctional cross-linker derivatized lysine side chains, and freecysteine residues. It was also found that that cross-linking of HBcAgsubunits leads to the formation of high molecular weight species ofundefined size which can not be resolved by SDS-polyacrylamide gelelectrophoresis.

When an antigen or antigenic determinant is linked to the non-naturalmolecular scaffold through a lysine residue, it may be advantageous toeither substitute or delete one or both of the naturally resident lysineresidues located at positions corresponding to positions 7 and 96 in SEQID NO:1, as well as other lysine residues present in HBcAg variants. Theelimination of these lysine residues results in the removal of bindingsites for antigens or antigenic determinants which could disrupt theordered array and should improve the quality and uniformity of the finalvaccine composition.

In many instances, when both of the naturally resident lysine residuesat positions corresponding to positions 7 and 96 in SEQ ID NO:1 areeliminated, another lysine will be introduced into the HBcAg as anattachment site for an antigen or antigenic determinant. Methods forinserting such a lysine residue are set out, for example, in Example 23of WO 02/056905, which is incorporated hereby by reference in itsentirety. It will often be advantageous to introduce a lysine residueinto the HBcAg when, for example, both of the naturally resident lysineresidues at positions corresponding to positions 7 and 96 in SEQ ID NO:1are altered and one seeks to attach the antigen or antigenic determinantto the non-natural molecular scaffold using a heterobifunctionalcross-linking agent.

The C-terminus of the HBcAg has been shown to direct nuclearlocalization of this protein (Eckhardt et al., J. Virol. 65:575-582(1991).) Further, this region of the protein is also believed to conferupon the HBcAg the ability to bind nucleic acids.

In some embodiments, vaccine compositions of the invention will containHBcAgs which have nucleic acid binding activity (e.g., which contain anaturally resident HBcAg nucleic acid binding domain). HBcAgs containingone or more nucleic acid binding domains are useful for preparingvaccine compositions which exhibit enhanced T-cell stimulatory activity.Thus, the vaccine compositions of the invention include compositionswhich contain HBcAgs having nucleic acid binding activity. Furtherincluded are vaccine compositions, as well as the use of suchcompositions in vaccination protocols, where HBcAgs are bound to nucleicacids. These HBcAgs may bind to the nucleic acids prior toadministration to an individual or may bind the nucleic acids afteradministration.

Further HBcAgs suitable for use in the practice of the present inventioninclude N- and C-terminal truncation mutants, and muteins whose aminoacid sequences comprises or alternatively consists of, amino acidsequences which are at least about 80%, about 85%, about 90%, about 95%,about 97%, or about 99% identical to the above described truncationmutants.

As discussed above, in certain embodiments of the invention, a lysineresidue is introduced as a first attachment site into a polypeptidewhich forms the non-natural molecular scaffold. In preferredembodiments, vaccine compositions of the invention are prepared using aHBcAg comprising, or alternatively consisting of, amino acids 1-144 oramino acids 1-149 or amino acids 1-185 of SEQ ID NO:1 which is modifiedso that the amino acids corresponding to positions 79 and 80 arereplaced with a peptide having the amino acid sequence ofGly-Gly-Lys-Gly-Gly (SEQ ID NO: 11) and the cysteine residues atpositions 48 and 107 are either deleted or substituted with anotheramino acid residue, while the cysteine at position 61 is left in place.

The invention further includes vaccine compositions comprising fragmentsof a HBcAg comprising, or alternatively consisting of, an amino acidsequence other than that shown in SEQ ID NO:1 from which a cysteineresidue not present at corresponding location in SEQ ID NO:1 has beendeleted.

Vaccine compositions of the invention may comprise mixtures of differentHBcAgs. Thus, these vaccine compositions may be composed of HBcAgs whichdiffer in amino acid sequence. For example, vaccine compositions couldbe prepared comprising a “wild-type” HBcAg and a modified HBcAg in whichone or more amino acid residues have been altered (e.g., deleted,inserted or substituted). The invention further includes vaccinecompositions where the non-natural molecular scaffold is prepared usinga HBcAg fused to another protein. As discussed above, one example ofsuch a fusion protein is a HBcAg/FOS fusion. Other examples of HBcAgfusion proteins suitable for use in vaccine compositions of theinvention include fusion proteins where an amino acid sequence has beenadded which aids in the formation and/or stabilization of HBcAg dimersand multimers. This additional amino acid sequence may be fused to theor C-terminus of the HBcAg. One example, of such a fusion protein is afusion of a HBcAg with the GCN4 helix region of Saccharomycescerevisiae, which forms homodimers via non-covalent interactions whichcan be used to prepare and stabilize HBcAg dimers and multimers.

In one embodiment, the invention provides vaccine compositions preparedusing HBcAg fusions proteins comprising a HBcAg, or fragment thereof,with a GCN4 polypeptide(PAALKRARNEAARRSRARKLQ-RMKQLEDKVEELLSKNYHLENEVARLKK (SEQ ID NO: 12))fused to the C-terminus. This GCN4 polypeptide may also be fused to theN-terminus of the HbcAg.

HBcAg/src homology 3 (SH3) domain fusion proteins could also be used toprepare vaccine compositions of the invention. SH3 domains arerelatively small domains found in a number of proteins which confer theability to interact with specific proline-rich sequences in proteinbinding partners (see McPherson, Cell Signal 11:229-238 (1999).HBcAg/SH3 fusion proteins could be used in several ways. First, the SH3domain could form a first attachment site which interacts with a secondattachment site of the antigen or antigenic determinant. Similarly, aproline rich amino acid sequence could be added to the HBcAg and used asa first attachment site for an SH3 domain second attachment site of anantigen or antigenic determinant. Second, the SH3 domain could associatewith proline rich regions introduced into HBcAgs. Thus, SH3 domains andproline rich SH3 interaction sites could be inserted into either thesame or different HBcAgs and used to form and stabilized dimers andmultimers of the invention.

As evidenced by the aforementioned example, one of skill in the artwould know how to form a molecular scaffold comprising core particlesand a first attachment site from HBcAg and HBcAg-derived muteins. Byapplication of art-known techniques and routine experimentation, itwould be understood by one of ordinary skill how other viruses could besimilarly used to construct a molecular scaffold.

As presented elsewhere herein, viral capsids may be used for (1) thepresentation or haptens and (2) the preparation of vaccine compositionsof the invention. Particularly, useful in the practice of the inventionare viral capsid proteins, also referred to herein as “coat proteins,”which upon expression form capsids or capsid-like structures. Thus,these capsid proteins can form core particles and non-natural molecularscaffolds. Generally, these capsids or capsid-like structures formordered and repetitive arrays which can be used for the presentation ofhaptens determinants and the preparation of vaccine compositions of theinvention.

One or more (e.g., one, two, three, four, five, etc.) haptens may beattached by any number of means to one or more (e.g., one, two, three,four, five, etc.) proteins which form viral capsids or capsid-likestructures (e.g., bacteriophage coat proteins), as well as otherproteins. For example, haptens may be attached to core particles usingfirst and second attachment sites. Further, one or more (e.g., one, two,three, four, five, etc.) heterobifunctional crosslinkers can be used toattach haptens determinants to one or more proteins which form viralcapsids or capsid-like structures.

Viral capsid proteins, or fragments thereof may be used, for example, toprepare core particles and vaccine compositions of the invention.Bacteriophage Qβ coat proteins, for example, can be expressedrecombinantly in E. coli. Further, upon such expression these proteinsspontaneously form capsids, which are virus-like particles.Additionally, these capsids form ordered and repetitive antigen arrayswhich can be used for hapten presentation and the preparation of vaccinecompositions. As described below in Example 1, bacteriophage Qβ coatproteins can be used to prepare vaccine compositions which elicitimmunological responses to haptens.

In a preferred embodiment, the virus-like particle comprises, consistsessentially of, or alternatively consists of recombinant proteins, orfragments thereof, of a RNA-phage. Preferably, the RNA-phage is selectedfrom the group consisting of a) bacteriophage Qβ; b) bacteriophage R17;c) bacteriophage fr; d) bacteriophage GA; e) bacteriophage SP; f)bacteriophage MS2; g) bacteriophage M11; h) bacteriophage MX1; i)bacteriophage NL95; k) bacteriophage f2; l) bacteriophage PP7, and m)bacteriophage AP205.

In another preferred embodiment of the present invention, the virus-likeparticle comprises, or alternatively consists essentially of, oralternatively consists of recombinant proteins, or fragments thereof, ofthe RNA-bacteriophage Qβ or of the RNA-bacteriophage fr or of theRNA-bacteriophage AP205.

In a further preferred embodiment of the present invention, therecombinant proteins comprise, or alternatively consist essentially of,or alternatively consist of coat proteins of RNA phages.

Specific examples of bacteriophage coat proteins which can be used toprepare compositions of the invention include the coat proteins of RNAbacteriophages such as bacteriophage Qβ (SEQ ID NO:3, PIR Database,Accession No. VCBPQβ referring to Qβ CP; and SEQ ID NO: 4, Accession No.AAA16663 referring to Qβ A1 protein), bacteriophage R17 (SEQ ID NO: 24;PIR Accession No. VCBPR7), bacteriophage fr (SEQ ID NO: 25; PIRAccession No. VCBPFR), bacteriophage GA (SEQ ID NO: 26; GenBankAccession No. NP-040754), bacteriophage SP (SEQ ID NO: 27; GenBankAccession No. CAA30374 referring to SP CP and SEQ ID NO: 28, AccessionNo. NP 695026 referring to SP A1 protein), bacteriophage MS2 (SEQ ID NO:29; PIR Accession No. VCBPM2), bacteriophage M11 (SEQ ID NO: 30; GenBankAccession No. AAC06250), bacteriophage MX1 (SEQ ID NO: 31; GenBankAccession No. AAC14699), bacteriophage NL95 (SEQ ID NO: 32; GenBankAccession No. AAC14704), bacteriophage f2 (SEQ ID NO: 33; GenBankAccession No. P03611), bacteriophage PP7 (SEQ ID NO: 13), bacteriophageAP205 (SEQ ID NO:14). As one skilled in the art would recognize, anyprotein which forms capsids or capsid-like structures can be used forthe preparation of vaccine compositions of the invention. Furthermore,the A1 protein of bacteriophage Qβ (Genbank accession No. AAA16663 (SEQID NO: 4)) or C-terminal truncated forms missing as much as about 100,about 150 or about 180 amino acids from its C-terminus may beincorporated in a capsid assembly of Qβ coat proteins. The A1 proteinmay also be fused an element containing a first attachment site, forattachment of haptens containing a second attachment site. Generally,the percentage of A1 protein relative to Qβ CP in the capsid assemblywill be limited, in order to insure capsid formation.

Qβ coat protein has also been found to self-assemble into capsids whenexpressed in E. coli (Kozlovska T M. et al., GENE 137: 133-137 (1993)).The obtained capsids or virus-like particles showed an icosahedralphage-like capsid structure with a diameter of 25 nm and T=3 quasisymmetry. Further, the crystal structure of phage Qβ has been solved.The capsid contains 180 copies of the coat protein, which are linked incovalent pentamers and hexamers by disulfide bridges (Golmohammadi, R.et al., Structure 4: 543-5554 (1996)). Other RNA phage coat proteinshave also been shown to self-assemble upon expression in a bacterialhost (Kastelein, R A. et al., Gene 23: 245-254 (1983), Kozlovskaya, T M.et al., Dokl. Akad. Nauk SSSR 287: 452-455 (1986), Adhin, M R. et al.,Virology 170: 238-242 (1989), Ni, C Z., et al., Protein Sci. 5:2485-2493 (1996), Priano, C. et al., J. Mol. Biol. 249: 283-297 (1995)).The Qβ phage capsid contains, in addition to the coat protein, the socalled read-through protein A1 and the maturation protein A2. A1 isgenerated by suppression at the UGA stop codon and has a length of 329aa. The capsid of phage Qβ recombinant coat protein used in theinvention is devoid of the A2 lysis protein, and contains RNA from thehost. The coat protein of RNA phages is an RNA binding protein, andinteracts with the stem loop of the ribosomal binding site of thereplicase gene acting as a translational repressor during the life cycleof the virus. The sequence and structural elements of the interactionare known (Witherell, G W. & Uhlenbeck, O C. Biochemistry 28: 71-76(1989); Lim F. et al., J. Biol. Chem. 271: 31839-31845 (1996)). The stemloop and RNA in general are known to be involved in the virus assembly(Golmohammadi, R. et al., Structure 4: 543-5554 (1996).)

Upon expression in E. coli, the N-terminal methionine of Qβ coat proteinis usually removed, as we observed by N-terminal Edman sequencing asdescribed in Stoll, E. et al. J. Biol. Chem. 252:990-993 (1977). VLPcomposed from Qβ coat proteins where the N-terminal methionine has notbeen removed, or VLPs comprising a mixture of Qβ coat proteins where theN-terminal methionine is either cleaved or present are also within thescope of the present invention.

Further preferred virus-like particles of RNA-phages, in particular ofQβ, in accordance of this invention are disclosed in WO 02/056905, thedisclosure of which is herewith incorporated by reference in itsentirety.

Further RNA phage coat proteins have also been shown to self-assembleupon expression in a bacterial host (Kastelein, R A. et al., Gene 23:245-254 (1983), Kozlovskaya, T M. et al., Dokl. Akad. Nauk SSSR 287:452-455 (1986), Adhin, M R. et al., Virology 170: 238-242 (1989), Ni, CZ., et al., Protein Sci. 5: 2485-2493 (1996), Priano, C. et al., J. Mol.Biol. 249: 283-297 (1995)). The Qβ phage capsid contains, in addition tothe coat protein, the so called read-through protein A1 and thematuration protein A2. A1 is generated by suppression at the UGA stopcodon and has a length of 329 aa. The capsid of phage Qβ recombinantcoat protein used in the invention is devoid of the A2 lysis protein,and contains RNA from the host. The coat protein of RNA phages is an RNAbinding protein, and interacts with the stem loop of the ribosomalbinding site of the replicase gene acting as a translational repressorduring the life cycle of the virus. The sequence and structural elementsof the interaction are known (Witherell, G W. & Uhlenbeck, O C.Biochemistry 28: 71-76 (1989); Lim F. et al., J. Biol. Chem. 271:31839-31845 (1996)). The stem loop and RNA in general are known to beinvolved in the virus assembly (Golmohammadi, R. et al., Structure 4:543-5554 (1996)).

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively consists essentially ofor alternatively consists of recombinant proteins, or fragments thereofof a RNA-phage, wherein the recombinant proteins comprise, consistessentially of or alternatively consist of mutant coat proteins of RNAphages. In another preferred embodiment, the mutant coat proteins havebeen modified by removal of at least one lysine residue, more preferablyof at least two lysine residues, by way of substitution, or by additionof at least one lysine residue by way of substitution. Alternatively,the mutant coat proteins have been modified by deletion of at least onelysine residue, more preferably of at least two lysine residues, or byaddition of at least one lysine residue, more preferably of at least twolysine residues, by way of insertion.

In another preferred embodiment, the virus-like particle comprises,consists essentially of, or alternatively consists of recombinantproteins, or fragments thereof, of the RNA-bacteriophage Qβ, wherein therecombinant proteins comprise, consist essentially of, or alternativelyconsist of coat proteins having an amino acid sequence of SEQ ID NO:3,or a mixture of coat proteins having amino acid sequences of SEQ ID NO:3and of SEQ ID NO: 4 or mutants of SEQ ID NO: 4 and wherein theN-terminal methionine is preferably cleaved.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, consists essentially of or alternativelyconsists of recombinant proteins of Qβ, or fragments thereof, whereinthe recombinant proteins comprise, consist essentially of oralternatively consist of mutant Qβ coat proteins. In another preferredembodiment, these mutant coat proteins have been modified by removal ofat least one lysine residue by way of substitution, or by addition of atleast one lysine residue by way of substitution. Alternatively, thesemutant coat proteins have been modified by deletion of at least onelysine residue, or by addition of at least one lysine residue by way ofinsertion.

Four lysine residues are exposed on the surface of the capsid of Qβ coatprotein. Qβ mutants, for which exposed lysine residues are replaced byarginines can also be used for the present invention. The following Qβcoat protein mutants and mutant Qβ VLP's can, thus, be used in thepractice of the invention: “Qβ-240” (Lys13-Arg; SEQ ID NO:6), “Qβ-243”(Asn 10-Lys; SEQ ID NO:7), “Qβ-250” (Lys 2-Arg, Lys13-Arg; SEQ ID NO:8),“Qβ-251” (SEQ ID NO:9) and “Qβ-259” (Lys 2-Arg, Lys16-Arg; SEQ IDNO:10). Thus, in further preferred embodiment of the present invention,the virus-like particle comprises, consists essentially of oralternatively consists of recombinant proteins of mutant Qβ coatproteins, which comprise proteins having an amino acid sequence selectedfrom the group of a) the amino acid sequence of SEQ ID NO:6; b) theamino acid sequence of SEQ ID NO:7; c) the amino acid sequence of SEQ IDNO:8; d) the amino acid sequence of SEQ ID NO:9; and e) the amino acidsequence of SEQ ID NO:10. The construction, expression and purificationof the above indicated Qβ coat proteins, mutant Qβ coat protein VLP'sand capsids, respectively, are described in WO 02/056905. In particularis hereby referred to Example 18 of above mentioned application.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, consists essentially of or alternativelyconsists of recombinant proteins of Qβ, or fragments thereof, whereinthe recombinant proteins comprise, consist essentially of oralternatively consist of a mixture of either one of the foregoing Qβmutants and the corresponding A1 protein.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively essentially consists of,or alternatively consists of recombinant proteins, or fragments thereof,of RNA-phage AP205.

The AP205 genome consists of a maturation protein, a coat protein, areplicase and two open reading frames not present in related phages; alysis gene and an open reading frame playing a role in the translationof the maturation gene (Klovins, J., et al., J. Gen. Virol. 83: 1523-33(2002)). AP205 coat protein can be expressed from plasmid pAP283-58 (SEQID NO: 15), which is a derivative of pQb10 (Kozlovska, T. M. et al.,Gene 137:133-37 (1993)), and which contains an AP205 ribosomal bindingsite. Alternatively, AP205 coat protein may be cloned into pQb185,downstream of the ribosomal binding site present in the vector. Bothapproaches lead to expression of the protein and formation of capsids asdescribed in Example 10. Vectors pQb10 and pQb185 are vectors derivedfrom pGEM vector, and expression of the cloned genes in these vectors iscontrolled by the trp promoter (Kozlovska, T. M. et al., Gene 137:133-37(1993)). Plasmid pAP283-58 (SEQ ID NO:15) comprises a putative AP205ribosomal binding site in the following sequence, which is downstream ofthe XbaI site, and immediately upstream of the ATG start codon of theAP205 coat protein: tctagaATTTTCTGCGCACCCATCCCGGGTGGCGCCCAAAGTGAGGAAAATCACatg (SEQ ID NO: 16). The vector pQb185 comprises a ShineDelagarno sequence downstream from the XbaI site and upstream of thestart codon (tctagaTTAACCCAACGCGTAGGAG TCAGGCCatg (SEQ ID NO: 17), ShineDelagarno sequence underlined).

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively essentially consists of,or alternatively consists of recombinant coat proteins, or fragmentsthereof, of the RNA-phage AP205.

This preferred embodiment of the present invention, thus, comprisesAP205 coat proteins that form capsids. Such proteins are recombinantlyexpressed, or prepared from natural sources. AP205 coat proteinsproduced in bacteria spontaneously form capsids, as evidenced byElectron Microscopy (EM) and immunodiffusion. The structural propertiesof the capsid formed by the AP205 coat protein (SEQ ID NO: 14) and thoseformed by the coat protein of the AP205 RNA phage are nearlyindistinguishable when seen in EM. AP205 VLPs are highly immunogenic,and can be linked with antigens and/or antigenic determinants togenerate vaccine constructs displaying the antigens and/or antigenicdeterminants oriented in a repetitive manner. High titers are elicitedagainst the so displayed antigens showing that bound antigens and/orantigenic determinants are accessible for interacting with antibodymolecules and are immunogenic.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively essentially consists of,or alternatively consists of recombinant mutant coat proteins, orfragments thereof, of the RNA-phage AP205.

Assembly-competent mutant forms of AP205 VLPs, including AP205 coatprotein with the subsitution of proline at amino acid 5 to threonine(SEQ ID NO: 18), may also be used in the practice of the invention andleads to a further preferred embodiment of the invention. These VLPs,AP205 VLPs derived from natural sources, or AP205 viral particles, maybe bound to antigens to produce ordered repetitive arrays of theantigens in accordance with the present invention.

AP205 P5-T mutant coat protein can be expressed from plasmid pAP281-32(SEQ ID No. 19), which is derived directly from pQb185, and whichcontains the mutant AP205 coat protein gene instead of the Qβ coatprotein gene. Vectors for expression of the AP205 coat protein aretransfected into E. coli for expression of the AP205 coat protein.

Methods for expression of the coat protein and the mutant coat protein,respectively, leading to self-assembly into VLPs are described inExamples 9 and 10. Suitable E. coli strains include, but are not limitedto, E. coli K802, JM 109, RR1. Suitable vectors and strains andcombinations thereof can be identified by testing expression of the coatprotein and mutant coat protein, respectively, by SDS-PAGE and capsidformation and assembly by optionally first purifying the capsids by gelfiltration and subsequently testing them in an immunodiffusion assay(Ouchterlony test) or Electron Microscopy (Kozlovska, T. M. et al., Gene137:133-37 (1993)).

AP205 coat proteins expressed from the vectors pAP283-58 and pAP281-32may be devoid of the initial Methionine amino-acid, due to processing inthe cytoplasm of E. coli. Cleaved, uncleaved forms of AP205 VLP, ormixtures thereof are further preferred embodiments of the invention.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively essentially consists of,or alternatively consists of a mixture of recombinant coat proteins, orfragments thereof, of the RNA-phage AP205 and of recombinant mutant coatproteins, or fragments thereof, of the RNA-phage AP205.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively essentially consists of,or alternatively consists of fragments of recombinant coat proteins orrecombinant mutant coat proteins of the RNA-phage AP205.

Recombinant AP205 coat protein fragments capable of assembling into aVLP and a capsid, respectively are also useful in the practice of theinvention. These fragments may be generated by deletion, eitherinternally or at the termini of the coat protein and mutant coatprotein, respectively. Insertions in the coat protein and mutant coatprotein sequence or fusions of antigen sequences to the coat protein andmutant coat protein sequence, and compatible with assembly into a VLP,are further embodiments of the invention and lead to chimeric AP205 coatproteins, and particles, respectively. The outcome of insertions,deletions and fusions to the coat protein sequence and whether it iscompatible with assembly into a VLP can be determined by electronmicroscopy.

The particles formed by the AP205 coat protein, coat protein fragmentsand chimeric coat proteins described above, can be isolated in pure formby a combination of fractionation steps by precipitation and ofpurification steps by gel filtration using e.g. Sepharose CL-4B,Sepharose CL-2B, Sepharose CL-6B columns and combinations thereof. Othermethods of isolating virus-like particles are known in the art, and maybe used to isolate the virus-like particles (VLPs) of bacteriophageAP205. For example, the use of ultracentrifugation to isolate VLPs ofthe yeast retrotransposon Ty is described in U.S. Pat. No. 4,918,166,which is incorporated by reference herein in its entirety.

According to the present invention, one or more haptens may be attachedto one subunit of the capsid of RNA phages coat proteins. The ability tocouple several haptens per subunit of the capsid of the coat protein ofRNA phages and in particular of Qβ capsid allows for the generation of adense hapten array. Other viral capsids may be used for covalentattachment of haptens by way of chemical cross-linking, such for examplea HBcAg modified with a lysine residue in its major immunodominantregion (MIR; WO 00/32227). The distance between the spikes(corresponding to the MIR) of HBcAg is 50 Angströms (Wynne, S A. et al.,Mol. Cell 3: 771-780 (1999)), and therefore an hapten array withdistances shorter than 50 A cannot be generated

Capsids of Qβ coat protein display a defined number of lysine residueson their surface, with a defined topology with three lysine residuespointing towards the interior of the capsid and interacting with theRNA, and four other lysine residues exposed to the exterior of thecapsid. These defined properties favor the attachment of haptens to theexterior of the particle, and not to the interior where the lysineresidues interact with RNA. Capsids of other RNA phage coat proteinsalso have a defined number of lysine residues on their surface and adefined topology of these lysine residues. Another advantage of thecapsids derived from RNA phages is their high expression yield inbacteria, that allows the production of large quantities of material ataffordable cost.

Another feature of the capsid of Qβ coat protein is its stability. Qβsubunits are bound via disulfide bridges to each other, covalentlylinking the subunits. Qβ capsid protein also shows unusual resistance toorganic solvents and denaturing agents. Surprisingly, we have observedthat DMSO and acetonitrile concentrations as high as about 30%, andGuanidinium concentrations as high as about 1 M could be used withoutaffecting the stability or the ability to form hapten arrays of thecapsid. Thus, theses organic solvents may be used to couple hydrophobicmolecules, such as hormones, drugs and toxins. The high stability of thecapsid of Qβ coat protein is an important feature pertaining to its usefor immunization and vaccination of mammals and humans in particular.The resistance of the capsid to organic solvent allows the coupling ofhaptens not soluble in aqueous buffers.

Insertion of a cysteine residue into the N-terminal β-hairpin of thecoat protein of the RNA phage MS-2 has been described in the U.S. Pat.No. 5,698,424, the reference of which is incorporated herein in itsentirety. We note however, that the presence of an exposed free cysteineresidue in the capsid may lead to oligomerization of capsids by way ofdisulfide bridge formation. Other attachments contemplated in the aboveU.S. patent involve the formation of disulfide bridges between theantigen and the Qβ particle. Such attachments are labile tosulfhydryl-moiety containing molecules.

The reaction between an initial disulfide bridge formed with acysteine-residue on Qβ, and the antigen containing a free sulfhydrylresidue releases sulfhydryl containing species other than the hapten.These newly formes sulfhydryl containing species can react again withother disulfide bridges present on the particle, thus establishing anequilibrium. Upon reaction with the disulfide bridge formed on theparticle, the hapten may either form a disulfide bridge with thecysteine-residue from the particle, or with the cysteine -residue of theleaving group molecule which was forming the initial disulfide bridge onthe particle. Moreover, the other method of attachment described, usinga hetero-bifunctional cross-linker reacting with a cysteine on the Qβparticle on one side, and with a lysine residue on the antigen on theother side, may lead to a random orientation of the antigens on theparticle.

We further note that, in contrast to the capsid of the Qβ and Fr coatproteins, recombinant MS-2 described in U.S. Pat. No. 5,698,424 isessentially free of nucleic acids, while RNA is packaged inside the twocapsids mentioned above.

We describe new and inventive compositions allowing the formation ofrobust hapten arrays with variable hapten density. We show that veryhigh epitope density can be achieved by attaching haptens to VLPs.Futher, the density and spacing of haptens can be modified byalterations in the number and type of residues with suitable firstattachment sites. For example WO 02/056905 discloses a Qβ mutant coatprotein with additional lysine residues, suitable for obtaining higherdensity arrays than observed with wild type Qβ coat protein. Further,the aforesaid application also discloses compositions suitable forsimultaneous display of several hapten with appropriate spacing, andcompositions wherein the addition of accessory molecules, enhancingsolubility or modifiying the capsid in a suitable and desired way. OtherQβ coat protein mutants, forming capsids, which are virus-likeparticles, are disclosed in WO 02/056905 and are suitable for generatingcompositions of the invention. In particular, in occurrences wheresolubility of the hapten, and of the Qβ-hapten antigen array imposes alimit on the number of hapten residues that can be attached on the Qβvirus-like particle, mutants where lysine residues have been substitutedfor arginines, which do not have the same reactivity as lysine residues,can be used. When preparing these compositions, a high concentration ofhapten, or hapten modified to comprise a second attachment site, can beused to achieve complete reaction at the lysine residues on the mutantQβ virus-like particles, without generating potentially insolubleparticles with a higher number of attached haptens, as would be the casewhen using the wt Qβ virus-like particle.

The crystal structure of several RNA bacteriophages has been determined(Golmohammadi, R. et al., Structure 4:543-554 (1996)). Using suchinformation, one skilled in the art could readily identify surfaceexposed residues and modify bacteriophage coat proteins such that one ormore reactive amino acid residues can be inserted. Thus, one skilled inthe art could readily generate and identify modified forms ofbacteriophage coat proteins which can be used in the practice of theinvention. Thus, variants of proteins which form capsids or capsid-likestructures (e.g., coat proteins of bacteriophage Qβ, bacteriophage R17,bacteriophage fr, bacteriophage GA, bacteriophage SP, and bacteriophageMS2) can also be used to prepare vaccine compositions of the invention.

Although the sequence of the variants proteins discussed above willdiffer from their wild-type counterparts, these variant proteins willgenerally retain the ability to form capsids or capsid-like structures.Thus, the invention further includes vaccine compositions which containvariants of proteins which form capsids or capsid-like structures, aswell as methods for preparing such vaccine compositions, individualprotein subunits used to prepare such vaccine compositions. Thus,included within the scope of the invention are variant forms ofwild-type proteins which form ordered and repetitive hapten arrays(e.g., variants of proteins which form capsids or capsid-likestructures) and retain the ability to associate and form capsids orcapsid-like structures. Normally, C- an N-terminal trunction variantsretain the ability to form virus like particles. As a result, variantforms including deletion, addition, or subsitution, chimeric forms, andnaturally occuring variants are suitable components of the invention.

Bacterial Pili and pilin proteins. In another embodiment, the coreparticle of the invention comprises, preferably consists of, a bacterialpilus or pilus-like particle. The pilus particle comprises proteins,mutant proteins or fragments of pilin proteins produced by organismsincluding Escherichia coli; Haemophilus influenzae; Neisseriameningitidis; Neisseria gonorrhoeae; Caulobacter crescentus; Pseudomonasstutzeri; Pseudomonas aeruginosa; Salmonella spp; and Vibrio cholera. Ina preferred embodiment, the pilin proteins or fragments thereof areselected from the group consisting of: a) Type 1 pilin proteins; and b)P-pilin proteins. In another embodiment, the pilin proteins arerecombinant proteins, or the pilus or pilus-like particle comprises amixture of recombinant and non-recombinant proteins. In yet an otherembodiment, the pilus or pilus-like particle comprises type I pilinproteins or fragments thereof. In a further embodiment, the pilinproteins are mutant proteins, preferably proteins which have beenmodified by removal of at least one lysine residue by way ofsubstitution, by addition of at least one lysine residue by way ofsubstitution, by deletion of at least one lysine residue, or by additionof at least one lysine residue by way of insertion. In a preferredembodiment, the type I pilin proteins have an amino acid sequence as setforth in SEQ ID No: 2. In yet a further aspect, the invention provides acomposition comprising the conjugate of the invention wherein the coreparticle comprises, preferably consists of, a bacterial pilus orpilus-like particle, and a pharmaceutically acceptible carrier.

In other embodiments, a bacterial pilin, a subportion of a bacterialpilin, or a fusion protein which contains either a bacterial pilin orsubportion thereof is used to prepare vaccine compositions of theinvention. Examples of pilin proteins include pilins produced byEscherichia coli, Haemophilus influenzae, Neisseria meningitidis,Neisseria gonorrhoeae, Caulobacter crescentus, Pseudomonas stutzeri, andPseudomonas aeruginosa. The amino acid sequences of pilin proteinssuitable for use with the present invention include those set out inGenBank reports AJ000636, AJ132364, AF229646, AF051814, AF051815, andX00981, the entire disclosures of which are incorporated herein byreference.

Bacterial pilin proteins are generally processed to remove N-terminalleader sequences prior to export of the proteins into the bacterialperiplasm. Further, as one skilled in the art would recognize, bacterialpilin proteins used to prepare vaccine compositions of the inventionwill generally not have the naturally present leader sequence.

One specific example of a pilin protein suitable for use in the presentinvention is the P-pilin of E. coli (GenBank report AF237482). Anexample of a Type-1 E. coli pilin suitable for use with the invention isa pilin having the amino acid sequence set out in GenBank report P04128(SEQ ID NO:2), which is encoded by nucleic acid having the nucleotidesequence set out in GenBank report M27603. The entire disclosures ofthese GenBank reports are incorporated herein by reference. Again, themature form of the above referenced protein would generally be used toprepare vaccine compositions of the invention.

Bacterial pilins or pilin subportions suitable for use in the practiceof the present invention will generally be able to associate to formnon-natural molecular scaffolds. Methods for preparing pili andpilus-like structures in vitro are known in the art. Bullitt et al.,Proc. Natl. Acad. Sci. USA 93:12890-12895 (1996), for example, describethe in vitro reconstitution of E. coli P-pili subunits. Further, Eshdatet al (J. Bacteriol. 148:308-314 (1981)) describe methods suitable fordissociating Type-1 pili of E. coli and the reconstitution of pili. Inbrief, these methods are as follows: pili are dissociated by incubationat 37° C. in saturated guanidine hydrochloride. Pilin proteins are thenpurified by chromatography, after which pilin dimers are formed bydialysis against 5 mM tris(hydroxymethyl) aminomethane hydrochloride (pH8.0). Eshdat et al. also found that pilin dimers reassemble to form piliupon dialysis against the 5 mM tris(hydroxymethyl) aminomethane (pH 8.0)containing 5 mM MgCl₂.

Further, using, for example, conventional genetic engineering andprotein modification methods, pilin proteins may be modified to containa first attachment site to which a hapten is linked through a secondattachment site. Alternatively, haptens can be directly linked through asecond attachment site to amino acid residues which are naturallyresident in these proteins. These modified pilin proteins may then beused in immunizing compositions of the invention.

Bacterial pilin proteins used to prepare compositions of the inventionmay be modified in a manner similar to that described herein for HBcAg.For example, cysteine and lysine residues may be either deleted orsubstituted with other amino acid residues and first attachment sitesmay be added to these proteins. Further, pilin proteins may either beexpressed in modified form or may be chemically modified afterexpression. Similarly, intact pili may be harvested from bacteria andthen modified chemically.

In another embodiment, pili or pilus-like structures are harvested frombacteria (e.g., E. coli) and used to form vaccine compositions of theinvention. One example of pili suitable for preparing vaccinecompositions is the Type-1 pilus of E. coli, which is formed from pilinmonomers having the amino acid sequence set out in SEQ ID NO:2.

A number of methods for harvesting bacterial pili are known in the art.Bullitt and Makowski (Biophys. J. 74:623-632 (1998)), for example,describe a pilus purification method for harvesting P-pili from E. coli.According to this method, pili are sheared from hyperpiliated E. colicontaining a P-pilus plasmid and purified by cycles of solubilizationand MgCl₂ (1.0 M) precipitation. WO 02/056905 discloses harvesting andpurification of Type I pili from bacteria that naturally produce pili,or into which a vector has been introduced encoding the fim operonresponsible for pilus production.

Once harvested, pili or pilus-like structures may be modified in avariety of ways. For example, a first attachment site can be added tothe pili to which haptens may be attached through a second attachmentsite. In other words, bacterial pili or pilus-like structures can beharvested and modified to form non-natural molecular scaffolds.

Pili or pilus-like structures may also be modified by the attachment ofhaptens in the absence of a non-natural first attachment site. Forexample, antigens or antigenic determinants could be linked to naturallyoccurring cysteine resides or lysine residues. In such instances, thehigh order and repetitiveness of a naturally occurring amino acidresidue would guide the coupling of the antigens or antigenicdeterminants to the pili or pilus-like structures. For example, the pilior pilus-like structures could be linked to the second attachment sitesof the haptens using a heterobifunctional cross-linking agent.

When structures which are naturally synthesized by organisms (e.g.,pili) are used to prepare vaccine compositions of the invention, it willoften be advantageous to genetically engineer these organisms so thatthey produce structures having desirable characteristics. For example,when Type-1 pili of E. coli are used, the E. coli from which these piliare harvested may be modified so as to produce structures with specificcharacteristics. Examples of possible modifications of pilin proteinsinclude the insertion of one or more lysine residues, the deletion orsubstitution of one or more of the naturally resident lysine residues,and the deletion or substitution of one or more naturally residentcysteine residues (e.g., the cysteine residues at positions 44 and 84 inSEQ ID NO:2).

Further, additional modifications can be made to pilin genes whichresult in the expression products containing a first attachment siteother than a lysine residue (e.g., a FOS or JUN domain). Of course,suitable first attachment sites will generally be limited to those whichdo not prevent pilin proteins from forming pili or pilus-like structuressuitable for use in vaccine compositions of the invention. The abilityof recombinant pilin proteins to form pili may be determined by a numberof methods including electron microscopy.

Pilin genes which naturally reside in bacterial cells can be modified invivo (e.g., by homologous recombination) or pilin genes with particularcharacteristics can be inserted into these cells. For examples, pilingenes could be introduced into bacterial cells as a component of eithera replicable cloning vector or a vector which inserts into the bacterialchromosome. The inserted pilin genes may also be linked to expressionregulatory control sequences (e.g., a lac operator).

In most instances, the pili or pilus-like structures used in vaccinecompositions of the invention will be composed of single type of a pilinsubunit. However, the compositions of the invention also includevaccines comprising pili or pilus-like structures formed fromheterogenous pilin subunits. Pili or pilus-like structures composed ofidentical subunits will generally be used because they are expected toform structures which present highly ordered and repetitive antigenarrays.

Second attachment site. The preparation of molecular scaffolds withordered and repetitive arrays is provided by the present includingcompositions of capsids of RNA phage coat proteins with a high epitopedensity. The nature of the hapten, and nature and location of the secondattachment site on the hapten are important factors that may influencethe means available to construct conjugates of the invention, and theeffectiveness of those conjugates in inducing an immune response, as isunderstood by those of ordinary skill in the art.

A prerequisite for designing a second attachment site is the choice ofthe position at which it should be fused, inserted or generallyengineered and attached. A skilled artisan would know how to findguidance in selecting the position of the second attachment site, andmany factors may be considered relevant to this decision. The chemicaland/or crystal structure of the hapten may provide information on theavailability of domains or moieties on the molecule suitable forcoupling. A reactive moiety or domain's accessibility to solvent may bea limiting factor in the kinetics of chemical coupling to a firstattachment site. Groups suitable for coupling must be available, such assulthydryl residues. In general, in the case where immunization with ahapten is aimed at inhibiting the interaction of said hapten, which mayalso be a self-antigen, with its natural ligands, such as a receptor,the second attachment site will be added such that it allows generationof antibodies against the site of interaction with the natural ligands.Thus, the location of the second attachment site will selected such,that steric hindrance from the second attachment site or any linker oramino acid linker containing it, is avoided. In further embodiments, anantibody response directed at a site distinct from the interaction siteof the hapten, which can also be a self-antigen with its natural ligandis desired. In such embodiments, the second attachment site may beselected such that it prevents generation of antibodies against theinteraction site of said hapten with its natural ligands. Other factorsof consideration include the nature of the hapten, its biochemicalproperties, such as pI, charge distribution, further modification. Ingeneral, flexible linkers are favored.

Other criteria in selecting the position of the second attachment siteinclude the oligomerization state of the hapten, the site ofoligomerization, the presence of a cofactor, the chemical reactivity ofthe hapten, and the availability of experimental evidence disclosingsites in the hapten structure and sequence where modification of thehapten is compatible with the function of the hapten, or with thegeneration of antibodies recognizing said hapten and preferably,blocking hapten function.

One method of attachment of haptens comprising a polypeptide linker toVLPs, and in particular to capsids of RNA phage coat proteins is thelinking of a lysine residue on the surface of the capsid of RNA phagecoat proteins with a sulfhydryl group residue on the polypeptide linker,such as is found in cysteine residues. Similarly, free sulfhydryl groupson haptens may also be effective attachment sites. Where an oxidizedsulfhydryl groups must be in a reduced state in order to function as asecond attachment site, reduction of may be achieved with e.g. DTT, TCEPor β-mercaptoethanol.

In one preferred embodiment of the invention, the hapten is synthesizedin such a manner that it comprises a second attachment site which canreact with the lysine residue on the surface of the capsid of RNA phagecoat proteins.

According to the present invention, the epitope density on the capsid ofRNA phage coat proteins can be modulated by the choice of cross-linkerand other reaction conditions. For example, the cross-linkers Sulfo-GMBSand SMPH allow reaching high epitope density. Derivatization ispositively influenced by high concentration of reactants, andmanipulation of the reaction conditions can be used to control thenumber of antigens coupled to RNA phages capsid proteins, and inparticular to Qβ capsid protein. In addition, the number of firstattachment sites on the core particle is another factor affecting thedensity of the hapten array. In one embodiment of the present invention,we provide a Qβ mutant coat protein with additional lysine residues,suitable for obtaining higher density arrays.

Cross linking. Methods for linking the hapten to the core particle arewell within the working knowledge of the practitioner of ordinary skillin the art, and numerous references exist to aid such a practitioner(e.g., Sambrook, J. et al., eds., MOLECULAR CLONING, A LABORATORYMANUAL, 2nd. edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989); Ausubel, F. et al., eds., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John H. Wiley & Sons, Inc. (1997); Celis, J., ed.,CELL BIOLGY, Academic Press, 2^(nd) edition, (1998); Harlow, E. andLane, D., “Antibodies: A Laboratory Manual,” Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1988), all of which areincorporated herein by reference in their entirities.

Differing methods of achieving an association between the core particleand hapten are described herein and are further described in WO02/056905. Methods include the JUN and FOS leucine zipper proteindomains are utilized for the first and second attachment sites of theinvention, respectively.

Preferred embodiments of the invention comprise the coupling of thenon-natural molecular scaffold to the hapten by chemical cross-linking.There is a wide range of compounds which have been developed tofacilitate cross-linking of proteins/peptides or conjugation of proteinsto derivatized molecules, e.g., haptens. These include, but are notlimited, to carboxylic acid derived active esters (activated compounds),mixed anhydrides, acyl halides, acyl azides, alkyl halides,N-maleimides, imino esters, isocyanates and isothiocyanates, which areknown to those skilled in the art. These are capable of forming acovalent bond with a reactive group of a protein molecule. Dependingupon the activating group, the reactive group is the amino group of alysine residue on a protein molecule or a thiol group in a carrierprotein or a modified carrier protein molecule which, when reacted,result in amide, amine, thioether, amidine urea or thiourea bondformation. One skilled in the art may identify further suitableactivating groups, for example, in general reference texts such asChemistry of Protein Conjugation and Cross-Linking (Wong (1991) CRCPress, Inc., Boca Raton, Fla.). Most reagents react preferentially withlysine side chain groups.

In some embodiments, the antigen is attached to the core particle by wayof chemical cross-linking, using a heterobifunctional cross-linker.Several hetero-bifunctional cross-linkers are known in the art. In oneembodiment, the hetero-bifunctional cross-linker contains a functionalgroup which can react with the side-chain amino group of lysine residuesof the core particle, and a functional group which can react with acysteine residue or sulfhydryl group present on the hapten, madeavailable for reaction by reduction, or engineered or attached on thehapten and optionally also made available for reaction by reduction. Thefirst step of the procedure, called the derivatization, is the reactionof the core particle with the cross-linker. The product of this reactionis an activated core particle, also called activated carrier. In thesecond step, unreacted cross-linker is removed using usual methods suchas gel filtration or dialysis. In the third step, the antigen is reactedwith the activated core particle, and this step is called the couplingstep. Unreacted antigen may be optionally removed in a fourth step.

In an alternative embodiment, the hapten is derivatized with an activemoiety suitable for cross linking to the first attachment site,generating an activated hapten. Such derivatization may occur on anisolated hapten or via a chemical synthesis. The activated hapten isthen reacted with the core particle such that coupling occurs.

Several hetero-bifunctional cross-linkers are known in the art. Theseinclude the cross-linkers SMPH (Pierce), Sulfo-MBS, Sulfo-EMCS,Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB, Sulfo-SMCC, SVSB, SIA and othercross-linkers available, for example from the Pierce Chemical Company(Rockford, Ill., USA), and having one functional group reactive towardsamino groups and one functional group reactive towards SH residues. Theabove mentioned cross-linkers all lead to formation of a thioetherlinkage. Another class of cross-linkers suitable in the practice of theinvention is characterized by the introduction of a disulfide linkagebetween the hapten and the core particle upon coupling. Cross-linkersbelonging to this class include for example SPDP and Sulfo-LC-SPDP(Pierce). The extent of derivatization of the core particle withcross-linker can be influenced by varying experimental conditions suchas the concentration of each of the reaction partners, the excess of onereagent over the other, the pH, the temperature and the ionic strength,as is well known from reaction theory in the field of organic chemistry.The degree of coupling, i.e. the amount of hapten per carrier can beadjusted by varying the experimental conditions described above to matchthe requirements of the vaccine. Solubility of the hapten may impose alimitation on the amount of antigen that can be coupled on each subunit,and in those cases where the obtained vaccine is insoluble, reducing theamount of antigens per subunit is beneficial.

In one specific embodiment the chemical agent is the heterobifunctionalcross-linking agent ε-maleimidocaproic acid N-hydroxysuccinimide ester(Tanimori et al., J. Pharm. Dyn. 4:812 (1981); Fujiwara et al., J.Immunol. Meth. 45:195 (1981)), which contains (1) a succinimide groupreactive with amino groups and (2) a maleimide group reactive with SHgroups. A heterologous protein or polypeptide of the first attachmentsite may be engineered to contain one or more lysine residues that willserve as a reactive moiety for the succinimide portion of theheterobifunctional cross-linking agent. Once chemically coupled to thelysine residues of the heterologous protein, the maleimide group of theheterobifunctional cross-linking agent will be available to react withthe SH group of a cysteine residue on the antigen or antigenicdeterminant. Antigen or antigenic determinant preparation in thisinstance may require the engineering of a sulfhydryl residue as thesecond attachment site so that it may be reacted to the free maleimidefunction on the cross-linking agent bound to the non-natural molecularscaffold first attachment sites. Thus, in such an instance, theheterobifunctional cross-linking agent binds to a first attachment siteof the non-natural molecular scaffold and connects the scaffold to asecond binding site of the antigen or antigenic determinant.

Other methods of coupling the hapten to the core particle includemethods wherein the hapten is cross-linked to the core particle usingcarbodiimide compounds. These include the carbodiimide EDC(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), which canoptionally also be used with N-hydroxy-succinimide NHS in the reaction.In one method, EDC is mixed with a hapten containing a free carboxylicacid, then added to the protein carrier. In other methods, the hapten isattached to the core particle using a homo-bifunctional cross-linkersuch as glutaraldehyde, DSG, BM[PEO]₄, BS³, (Pierce Chemical Company,Rockford, Ill., USA) or other known homo-bifunctional cross-linkers withfunctional groups reactive towards amine groups or carboxyl groups ofthe core particle.

Additional cross-linking methods and cross-linkers, suitable forattaching a hapten to a core particle and a virus-like particle,respectively, as well as guidance on performing the coupling reactionsand on the use of chemical cross-linkers and chemical cross-linkingprocedures can be found in Hermanson, G. T. in Bioconjugate Techniques,Academic Press Inc., San Diego, Calif., USA.

Further methods of binding the core particle to a hapten include methodswhere the core particle is biotinylated, and the hapten linked tostreptavidin, or methods wherein both the hapten and the core particleare biotinylated. In this case, the hapten may be first bound tostreptavidin or avidin by adjusting the ratio of antigen to streptavidinsuch that free binding sites are still available for binding of the coreparticle, which is added in the next step. Alternatively, all componentsmay be mixed in a “one pot” reaction. Other ligand-receptor pairs, wherea soluble form of the receptor and of the ligand is available, and arecapable of being cross-linked to the core particle or the hapten, may beused as binding agents for binding the hapten to the core particle.

Haptens. In one aspect, the invention provides ordered, repetitivehapten arrays suitable for immunization against haptens. Preferredhaptens are hormones, drugs and toxic compounds. More preferred aredrugs, especially addictive drugs. Immune responses against said drugs,hormones and toxic compounds may be used to protect an individual atrisk of exposure to said compounds, as therapy in an individual soexposed, or so as to prevent or treat addictions.

Representative hormones include, but are not limited to, progesterone,testosterone, estrogen, melanin stimulating hormone, cortisone, folliclestimulating hormone, adrenalin, and noradrenalin. Immune responsesagainst said hormones may be used in therapies against melanoma; cancersof the reproductive organs, such as breast, ovarian, uterine,testicular, and cervical cancers; and in conditions where alteration ofhormone levels is desired such as for contraception.

Representative toxic compounds include, but are not limited to, thenatural products of toxic plants, animals, and microorganisms; theseinclude but are not limited to aflatoxin, ciguautera toxin, andtetrodotoxin. Representative toxic compounds produced artificially, oras a result of metabolism include antibiotics (e.g vancomycin and thelike), anticancer compounds (eg vinblastine and the like) and chemicalwarfare agents (eg. botulinus toxin, sarin, tabun, soman, VX and thelike). An aspect of the invention includes the production of antibodiesagainst toxic metabolites of commonly used pharmaceutical agents, suchthat an individual may continue to receive the beneficial effects of apharmaceutical agents without side effects associated with toxicmetabolites.

The selection of antigens or antigenic determinants for compositions andmethods of treatment for drug addiction, in particular recreational drugaddiction, would be known to those skilled in the medical arts treatingsuch disorders. Representative examples of such antigens or antigenicdeterminants include, for example, opioids and morphine derivatives suchas codeine, fentanyl, heroin, morphium and opium; relaxants such asdiazepam; stimulants such as amphetamine, cocaine, MDMA(methylenedioxymethamphetamine), methamphetamine, methylphenidate andnicotine; hallucinogens such as PCP, LSD, mescaline and psilocybin;cannabinoids such as hashish and marijuana; as well as thedesipramine/imipramine class of drugs and thenortriptyline/amitriptyline class of drugs. Therapy for nicotineaddiction may also target nicotine metabolites including nornicotine andcotinine, both of which have longer half lives than nicotine, havepharmacologic and neuropharmacologic affects similar to nicotine and maybe addictive.

In the above embodiments, it is not necessary that the immunizing haptencomprising the entire molecule of hormone, drug, or toxin. Suitableimmune responses against the drug, hormone or toxin of interest may begenerated by the use of fragments of the drug, hormone or toxin, orrelated chemical structures.

The invention embodies different sites of linkage and means of linkageof the hapten to the carrier, and have been illustrated both earlier inthe invention, and by reference to the examples. Preferred sites andmeans of linkage may be determined on the basis of prior experience,theory and by routine experimentation.

Nicotine and nicotine metabolites. Immune responses suitable fornicotine may be generated by haptens coupled to the core particle eithervia the pyridine or pyrrolidine ring. In one embodiment,6-(carboxymethylureido)-(±)-nicotine (CMUNic) conjugate is synthesizedfrom 6-amino-(±)-nicotine, which is reacted with ethyl isocyanoacetateto form 6-(carboxyethylureido)-(±)-nicotine, and hydrolysis by lithiumhydroxide to form CMUNic as described (Hieda et al Int J Pharmacol22:809-819 (2000)), the reference to which is incorporated herein in itsentirety. The hapten is conjugated to the core particle via the terminalcarboxyl group, which may be activated using e.g.1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl. In anotherembodiment, 6-amino-(±)-nicotine is coupled to carrier proteins asdescribed by WO 99/61054, incorporated herein by reference in itsentirety.

In another embodiment of the present invention,trans-3′-aminomethylnicotine conjugate is prepared bytrans-3′-hydroxymethylnicotine alcohol via the the tosylate as describedby Cushman and Castignoli (J Org Chem 37:1268-1271 (1972)) the referenceto which is incorporated herein in its entirety. The hapten isconjugated to the core particle through a succinic acid linker using1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDAC) to activate thelinker's carboxylic acid group.

In a related embodiment, 3′-linkages to nicotine haptens are performedby first generating trans-3′-hydroxymethylnicotine which is reacted withsuccinic anhydride to yield the succinylatedhydroxymethylnicotine(O-succinyl-3′-hydroxymethyl-nicotine). Thisproduct is then mixed with EDAC and the core particle forcarbodiimide-activated coupling, as described by Langone and Van Vunakis(Methods Enzymol. 84:628-640 (1982)) the reference to which isincorporated herein in its entirety. In another embodiment,trans-4′-carboxycotinine is similarly activated with EDAC for couplingto a protein carrier.

In one embodiment, a nicotine hapten is coupled via the 1-positionNitrogen by conversion to the aminoethylpyridinium derivative,S-1-(b-aminoethyl)nicotinium chloride dihydrochloride, which is thencoupled to a core particle in the presence of1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonateas described Noguchi et al. (Biochem Biophys Res Comm. 83:83-86 (1978))the reference to which is incorporated herein in its entirety. In arelated embodiment, Cotinine is conjugated to core particles using thesame general approach, via formation of S-1-(b-aminoethyl)cotiniumchloride hydrochloride.

In one embodiment, a nicotine hapten is coupled via the 1′-position asdescribed by Isomura et al. (J. Org Chem 66:4115-4121 (2001), thereference to which is incorporated herein in its entirety, via formationof N[1-oxo-6-[(2S)-2-(3-pyridyl)-1-pyrrolidinyl]hexyl]-β-alanine. Thisactivated hapten is then coupled to a protein carrier. In three otherembodiments, conjugates are formed between the first attachment site ona protein core particle and the cotinine hapten4-oxo-4-[[6-[(5S)-2-oxo-5-(3-pyridinyl)-1-pyrrolidinyl]]hexyl]amino]-butanoicacid, or the nornicotine haptens(2S)-2-(3-pyridinyl)-1-pyrrolidinebutanoic acid phenylmethyl ester or(2R)-2-(3-pyridinyl)-1-pyrrolidinebutanoic acid phenylmethyl ester.

In one embodiment, cotinine 4′-carboxylic acid is covalently bound tocarriers at lysine as described by Bjerke et al. (J. Immunol. Methods,96, 239-246 (1987)) the reference to which is incorporated herein in itsentirety.

Nicotine haptens may be conjugated to carrier protein via a linker atthe 6-position of nicotine. Along these lines, the following haptens areused in embodiments of the present inventionN-succinyl-6-amino-(.±.)-nicotine;6-(.sigma.-aminocapramido)-(.±.)-nicotine and6-(.sigma.-aminocapramido)-(.±.)-nicotine, as described (Castro et al.Eur. J. Biochem., 104, 331-340 (1980); Castro et al. in Biochem.Biophys. Res. Commun. 67, 583-589 (1975); Castro et al. Res. CommunChem. Path. Pharm. 51, 393-404 (1986)), which is incorporated byreference herein in its entirety.

In other embodiments of the invention, nicotine haptens are conjugatedvia the 3′,4′, or 5′ position via succinylation of aminomethylnicotine,activation with EDC and subsequent mixture with the carrier, asdescribed by U.S. Pat. No. 6,232,082, the reference to which isincorporated herein in its entirety. In other embodiments, aminomethylnicotine is conjugated via polyglutamic acid-ADH to the core particle.In other embodiments, conjugates are formed from acetyl nicotine andaldehydo nicotine derivatized at the 3′,4′, or 5′ positions.

In other embodiments, hapten carrier conjugates comprise 5- and6-linkages of nicotine, including 5-(1-methyl-2-pyrrolidinyl)-2- or3-pyridinyl-conjugates and 5-(N-methyl-2-pyrrolidinyl)-2- or3-pyridinyl-conjugates. The construction of the haptens for theseconjugates is described in WO 99/61054, the reference to which isincorporated herein in its entirety. In other embodiments, 5- and6-amino nicotine are utilized as starting materials that are furtherderivatized at the amino group to add, typically, carbon chains thatterminate in a suitably reactive group including amines and carboxylicacids. These haptens are then suitable for conjugation to core particlesof the present invention. In other embodiments, 5- or 6-bromonicotine isused as a suitable starting material for reaction with alkynes leadingto the addition of unsaturated carbon groups with a chain whichterminate with moeities suitable for coupling, including amines andcarboxylic acids, that allow conjugation to the core particle.

Other embodiments of the present invention comprise conjugatescomprising nicotine haptens conjugated at the 1, 2, 4, 5, 6, or 1′positions of the nicotine, as described by Swain et al. (WO 98/14216),herein incorporated by reference in its entirety.

Other embodiments of the present invention comprise conjugatescomprising nicotine haptens as described by Janda et al. (WO 02/058635).

Further embodiments comprise conformationally constrained nicotinehaptens as described in Meijler et al. (J. Am. Chem. Soc, 2003, 125,7164-7165).

Cocaine and related drugs. The present invention provides conjugates,compositions and methods comprising cocaine conjugated to a coreparticle. In one group of embodiments, the diazonium salts of benzoylcocaine and benzoyl ecognine are coupled to carrier proteins. In otherembodiements the para-imino ester derivatives of cocaine and norcocaineare conjugated to core particles. Haptens suitable for these embodimentsare described in U.S. Pat. Nos. 3,88,866, 4,123,431 and 4,197,237 thereferences to which are incorporated herein in their entireties.Conjugates of cocaine using the the para-position of the the phenyl ringof various cocaine derivatives show increased stability to hydrolysis bythe introduction of an amide bond.

Other embodiments of the present invention comprise cocaine haptensdescribed by U.S. Pat. No. 5,876,727, the reference to which isincorporated herein in its entirety.

In one embodiment, precursors of the conjugates of the instant inventionare synthesized by acylating ecgonine methyl ester with bromoacetylbromide in DMF in the presence of two equivalents ofdiisopropylethylamine. The product is then coupled to the thiol group ofa thiolated carrier protein to obtain a conjugate.

In another embodiment, precursors of the conjugates of the instantinvention are synthesized by succinylating ecgonine methyl ester withsuccinic anhydride in DMF in the presence of one equivalent oftriethylamine. The product is then coupled to the amino group of alysine residue of a carrier protein to obtain a conjugate. In oneembodiment, precursors of the conjugates of the instant invention aresynthesized by reacting norcocaine with succinic anhydride in methylenechloride in the presence of two equivalents of triethylamine. In otheremobidments precursors of the conjugates of the instant invention aresynthesized by reacting a solution of norcocaine monoactivated succinicacid and triethylamine to form succinylated norcocaine. In either case,the resulting succinyl norocaine consists of a mixture of at least twoisomers, namely the exo and endo forms of the succinyl group. In theseembodiments succinyl norocaine is then be coupled to the .epsilon.-aminogroup of a lysine residue of a carrier protein using EDC to obtain aconjugate. In an alternative embodiment, the coupling reaction iscarried out using a pre-activated succinylated norcocaine derivative.That is, the intermediate can be isolated and characterized. Thepre-activated succinylated norcocaine derivative is synthesized byreacting 4-hydroxy-3-nitrobenzene sulfonic acid sodium salt withsuccinylated norcocaine in the presence of dicyclohexylcarbodiimide(DCC) and DMF. The product is conjugated to the amino group of a lysineresidue of a carrier protein to obtain a conjugate.

In one alternative embodiment, compounds of the present invention aresynthesized by reacting succinylated norcocaine with N-hydroxysuccimidein the presence of ethyl chloroformate, N-methylmorpholine (NMM) andDMF. The product is then coupled to the amino group of a lysine residueof a carrier protein to obtain a conjugate.

In one embodiment, compounds of the instant invention are synthesized byreacting thionyl chloride with succinylated norcocaine. The product isthen conjugated to a carrier protein to obtain a conjugate. In anotherembodiment, compounds of the instant invention are synthesized byreacting succinylated norcocaine with HATU in DMF anddiisopropylethylamine as outlined by Carpino ((1993) J. Am. Chem. Soc.115:4397-4398) the reference to which is incorporated herein in itsentirety. The product is added to an aqueous solution containing thecarrier protein to obtain a conjugate.

In another embodiment, compounds of the invention are synthesized byreacting succinylated norcocaine with PyBroP in DMF anddiisopropylethylamine. The product is added to an aqueous solutioncontaining the carrier protein to obtain a conjugate. In a relatedembodiment the carrier protein is succinylated with succinic anhydridein borate buffer. The product is then coupled to norcocaine in thepresence of EDC to obtain a conjugate.

In another embodiment, reduction of the free acid of coacaine in benzoylecgonine to its corresponding primary alcohol, is achieved usingborane-dimethylsulfide complex. The alcohol is reacted with succinicanhydride in DMF, the product of which is then conjugated to the freeamino acid group of a carrier protein in the presence of EDC to obtain aconjugate.

In another embodiment, compounds of the instant invention aresynthesized by conjugating benzoyl ecgonine to the amino group of alysine residue of a carrier protein in the presence of EDC to obtain aconjugate.

In one embodiment, the precursor of the conjugates is synthesized byacylating racemic nornicotine with succinic anhydride in methylenechloride in the presence of two equivalents of diisopropylethylamine.The product of this reaction is then coupled to the lysine residue of acarrier protein using HATU to obtain the conjugate. In anotherembodiment, selectively alkylating the pyridine nitrogen in(S)-(−)-nicotine in anhydrous methanol, with ethyl 3-bromobutyrate,5-bromovaleric acid, 6-bromohexanoic acid or 8-bromooctanoic acid yieldproducts suitable for conjugation to a carrier protein using HATU.

Compositions, Vaccines, and the Administration Thereof, and Methods ofTreatment

As discussed herein, the invention provides compositions which may beused for preventing and/or treating diseases or conditions. Theinvention further provides vaccination methods for preventing and/ortreating diseases or conditions in individuals. In a preferredembodiment, compositions stimulate an immune response leading to theproduction of immune molecules, including antibodies, that bind toorganic molecules. The invention further provides vaccination methodsfor preventing and/or treating diseases or conditions in individuals.

The nature or type of immune response is not a limiting factor of thisdisclosure. The desired outcome of a therapeutic or prophylactic immuneresponse may vary according to the disease, according to principles wellknown in the art. For example, a vaccine against an inhaled drug (egnicotine, cocaine) may be designed to induce high titres of serum IgGand also of secreted sIgA antibodies in the respiratory epithelium, thusbinding nicotine both in the respiratory tract and within thebloodstream. By comparison, titres of sIgA antibodies are presumablyless relevant when targeting an injected drug of abuse (eg heroin).However, a vaccination methodology against an injected drug of abusethat results in high serum titres as well as sIgA will nontheless beeffective, so long as serum titres are sufficient.

The invention comprises vaccines sufficient to cure or prevent a diseaseor condition or addiction. The invention further comprises vaccines thatreduce the number, severity or duration of symptoms; and vaccinecompositions effective in reducing the number of individuals in apopulation with symptoms. The invention comprises compositions witheffects upon the immune system that may aid in the treatment of adisease, as one facet in an overall therapeutic intervention against adisease. Given the notably complex nature of addiction, the inventioncomprises compositions that aid in therapy against drug addiction butare accompanied by psychiatric, behavioural, social and legalinterventions.

Furthermore, it may be desired to stimulate different types of immuneresponse depending on the disease, and according to principles known inthe art. It is well known, for example, that some immune responses aremore appropriate for a particular antigen than other immune responses.Some immune responses are, indeed, inappropriate and can causepathology, such as pathologic inflammation.

The nature of the immune response can be affected by the nature of theantigen, route of introduction into the body, dose, dosage regimen,repetitive nature of the antigen, host background, and signallingfactors of the immune system. Such knowledge is well known in the art.As such, an immune response may be tailored by the application of bothart known theory and routine experimentation.

Furthermore, the invention embodies the use of differing core particlesduring the course of vaccination against a drug or drugs. Individualswho develop strong immune responses against core particles such as e.g.pili, may be immunized with compositions comprising the same hapten butdiffering in core particle.

While not wishing to be bound by theory or any particular mechanisticexplanation for operation of the present invention, the conjugates ofthe present invention provide particular novel and surprising advantagesas components of pharmaceutical compositions to generate an immuneresponse, and particularly as vaccines. Other carriers known in the artincluding BSA, keyhole limpet hemocyanin, tetanus toxoid, bacterialoutermembrane proteins, cholera toxin, and Pseudomonas aeruginosaExotoxin A may be inappropriate for use in an individual, and inparticular a human. The aforementioned carriers may induce allergicreactions, or stimulate pathologic immune responses (for example,cholera toxin, KLH, BSA). The aforementioned carriers may require thepresence of adjuvants such as complete Freunds adjuvant, now consideredinappropriate for use in humans. A number of the carriers may becomponents of current vaccines (for example, tetanus toxiod, choleratoxin, Exotoxin A). As such, an individual may possess a high level ofpre-existing immunity to these carriers, such that immunization with anantigen-carrier conjugate will induce a relatively greater immuneresponse to the carrier than to the novel antigen. For these reasons,individually or as a whole, the conjugates and compositions of thepresent invention represent a useful improvement over theabove-described carrier proteins. The preseent invention demonstratesthe use of Nicotine-Qβ VLP conjugate composition to stimulate an immuneresponse against nicotine without the use of complete Freund's adjuvantand without evidence of pathologic immune responses.

In the use of the embodiments of the invention, haptens conjugated tocore particles can be taken up by antigen presenting cells and therebystimulate T-cell help to induce immune response. T helper cell responsescan be divided into type 1 (T_(H)1) and type 2 (T_(H)2) T helper cellresponses (Romagnani, Immunol. Today 18:263-266 (1997)). T_(H)1 cellssecrete interferon-gamma and other cytokines which trigger B cells toproduce IgG1-3 antibodies. In contrast, a critical cytokine produced byT_(H)2 cells is IL-4, which drived B cells to produce IgG4 and IgE. Inmany experimental systems, the development of T_(H)1 and T_(H)2responses is mutually exclusive since T_(H)1 cells suppress theinduction of T_(H)2 cells and vice versa. Thus, antigens that trigger astrong T_(H)1 response simultaneously suppress the development of T_(H)2responses and hence the production of IgE antibodies. Interestingly,virtually all viruses induce a T_(H)1 response in the host and fail totrigger the production of IgE antibodies (Coutelier et al., J. Exp. Med.165:64-69 (1987)). Antibodies of the IgE isotype are importantcomponents in allergic reactions. Mast cells bind IgE antibodies ontheir surface and release histamines and other mediators of allergicresponse upon binding of specific antigen to the IgE molecules bound onthe mast cell surface. The isotype pattern typical of T_(H)1 responsesis not restricted to live viruses but has also been observed forinactivated or recombinant viral particles (Lo-Man et al., Eur. J.Immunol. 28:1401-1407 (1998)). Thus, by using the processes of theinvention (e.g., AlphaVaccine Technology), viral particles can bedecorated with various hapten and used for immunization. Due to theresulting “viral structure” of the hapten, a T_(H)1 response will beelicited, “protective” IgG1-3 antibodies will be produced, and theproduction of IgE antibodies which cause allergic reactions will beprevented. Thus, the invention embodies compositions capable of inducingpreferred immune responses, notably T_(H)1 type responses. Futher, theinvention embodies the use of compositions of the invention to counterallergic reactions induced by alternative vaccines against haptens ofinterest.

A further advantageous feature of the present invention is that haptensmay be presented on the in regular, repetitive arrays that are able toinduce efficient immune responses both with and without T-cell help.This feature of the invention is particularly advantageous.

Unlike isolated proteins, viruses induce prompt and efficient immuneresponses in the absence of any adjuvants both with and without T-cellhelp (Bachmann & Zinkernagel, Ann. Rev. Immunol: 15:235-270 (1997)).Although viruses often consist of few proteins, they are able to triggermuch stronger immune responses than their isolated components. ForB-cell responses, it is known that one crucial factor for theimmunogenicity of viruses is the repetitiveness and order of surfaceepitopes. Many viruses exhibit a quasi-crystalline surface that displaysa regular array of epitopes which efficiently crosslinksepitope-specific immunoglobulins on B cells (Bachmann & Zinkernagel,Immunol. Today 17:553-558 (1996)). This crosslinking of surfaceimmunoglobulins on B cells is a strong activation signal that directlyinduces cell-cycle progression and the production of IgM antibodies.Further, such triggered B cells are able to activate T helper cells,which in turn induce a switch from IgM to IgG antibody production in Bcells and the generation of long-lived B cell memory—the goal of anyvaccination (Bachmann & Zinkernagel, Ann. Rev. Immunol. 15:235-270(1997)). The present invention provides one way to improve theefficiency of vaccination by increasing the degree of repetitiveness ofthe hapten to be used for immunization, through binding of the hapten tothe core particles. As previously noted, the invention provides forcompositions comprising core particle modified to alter the number andor arrangement of the first attachment sites.

As will be understood by one of ordinary skill in the art, whenconjugates of the present invention are administered to an individual,they may be in a composition which contains salts, buffers, adjuvantsand other substances, excipients or carriers which are desirable forimproving the efficacy of the composition. Examples of materialssuitable, or acceptible, for use in preparing pharmaceuticalcompositions are provided in numerous sources including REMINGTON'SPHARMACEUTICAL SCIENCES (Osol, A, ed., Mack Publishing Co., (1990)).

Compositions of the invention are said to be “pharmacologicallyacceptable” if their administration can be tolerated by a recipientindividual. Further, the compositions of the invention will beadministered in a “therapeutically effective amount” (i.e., an amountthat produces a desired physiological effect).

The compositions of the present invention may be administered by variousmethods known in the art, but will normally be administered byinjection, infusion, inhalation, oral administration, or other suitablephysical methods. The compositions may alternatively be administeredintramuscularly, intravenously, transmucosally, transdermally orsubcutaneously. Components of compositions for administration includesterile aqueous (e.g., physiological saline) or non-aqueous solutionsand suspensions. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oils such as olive oil, and injectableorganic esters such as ethyl oleate. Carriers or occlusive dressings canbe used to increase skin permeability and enhance antigen absorption.

In one specific embodiment, a human with nicotine addiction is immunizedwith 5 to 500 μg, preferably 25 to 200 μg, more preferably 50 to 100 μg,most preferably 100 μg of Nic-Qβ conjugate, with boosts at 3 weeks andagain at 6 weeks, more preferably with boosts at 4 weeks and again at 8weeks. Routes of immunizations can comprise intramuscular, subcutaneous,intradermal, transdermal, or intravenous injections. Two weeks afterimmunization, the immune response is monitored with kits as describedelsewhere herein. The resulting immune response is specific for nicotineand comprises high serum IgG, and is sufficient to inhibit nicotineuptake into the brain. The resulting immune response is long lasting andthus the individual does not experience pleasurable effects fromnicotine, and ceases nicotine use. Those skilled in the art will knowfrom the measured immune response whether additional immunizations willbe needed to maintain nicotine specific IgG levels. In an alternativeembodiment of the present invention the nicotine-hapten carrierconjugates of the invention are administered by intranasal vaccination.This type of administration leads to high antibody titers encompassingIgA as indicated in the examples.

In a further embodiment of the invention, a pharmaceutical compositionis provided for treating nicotine addiction, palliating nicotinewithdrawal symptoms, facilitating smoking cessation or preventingrelapse comprising a therapeutically effective combination of thevaccine composition of the invention and an additional agent. In oneembodiment, the additional agent is selected from the group consistingof: anti-depressant; nicotine receptor modulator; cannabinoid receptorantagonist; opioid receptor antagonist; monoamine oxidase inhibitor;anxiolytic or any combination of these agents. Preferably, theadditional agent is an anti-depressant selected from the groupconsisting of bupropion, doxepin, desipramine, clomipramine, imipramine,nortriptyline, amitriptyline, protriptyline, trimipramine, fluoxetine,fluvoxamine, paroxetine, sertraline, phenelzine, tranylcypromine,amoxapine, maprotiline, trazodone, venlafaxine, mirtazapine, theirpharmaceutically active salts and their optical isomers. In a verypreferred embodiment, the anti-depressant is either bupropion or apharmaceutically acceptable salt thereof, or nortriptyline or apharmaceutically acceptable salt thereof.

In another embodiment, the additional agent is a nicotine receptormodulator selected from the group consisting of mecamylamine, SSR591813,amantadine, pempidine, dihydro-beta-erythroidine, hexamethonium,erysodine, chlorisondamine, trimethaphan camsylate, tubocurarinechloride, d-tubocurarine, varenicline, their pharmaceutically acceptablesalts and their optical isomers. In a very preferred embodiment, thenicotine receptor modulator is mecamylamine or a pharmaceuticallyacceptable salt thereof. In another preferred embodiment, the nicotinereceptor modulator is varenicline or a pharmaceutically acceptable saltthereof.

In one embodiment, the present invention comprises a method of treatingtobacco addiction or nicotine addiction, palliating nicotine withdrawalsymptoms, preventing relapse or facilitating smoking cessationcomprising the step of administering to a patient the vaccinecomposition of the invention and an additional agent. In a preferredembodiment, the vaccine composition is administered intranasally,orally, subcutaneously, transdermally, intramuscularly or intravenously,and wherein said additional agent is administered orally or via atransdermal patch. In a more preferred embodiment, the vaccinecomposition of the invention comprisesO-succinyl-3′-hydroxymethyl-nicotine conjugated to Qβ virus likeparticle.

Anti-depressants, nicotine receptor agonists and antagonists,cannabinoid and opioid receptor antagonists, monoamine oxidaseinhibitors and anxiolytics are able to relieve certain symptoms duringsmoking cessation such as withdrawal, craving, depression, irritability,anergia, amotivation, appetite changes, nausea and hypersomnia. Theymainly act directly on receptors in the brain. Furthermore, weightincrease upon smoking cessation is a major concern for a number ofpeople. Vaccination inhibits the uptake of the nicotine into the brainand thus inhibits its rewarding effects. It does not inhibit withdrawalsymptoms but inhibits the reinforcement of nicotine addiction upon aslip. Therefore, a combination of vaccination and the use ofanti-depressants, nicotine receptor antagonists, cannabinoid receptorantagonists, monoamine oxidase inhibitors and anxiolytics and furtherdrugs inhibiting weight gain is beneficial for aid in smoking cessationand relapse prevention.

Anti-depressants are used to treat symptoms of nicotine withdrawal andaid smoking cessation. One such anti-depressant is bupropion and asustained-release formulation of bupropion HCl under the tradename Zybanis marketed as an aid for smoking cessation. The mechanism of action ofbupropion is presumed to involve inhibition of neural re-uptake ofdopamine and/or norepinephrine. As dopamine has been associated with therewarding, effects of addictive substances, such as nicotine, inhibitionof the norepinephrine uptake was contemplated to induce a decrease ofwithdrawal symptoms. Methods to produce bupropion and pharmaceuticallyacceptable salts thereof are disclosed in U.S. Pat. Nos. 3,819,706 and3,885,046. Methods to produce optically pure (+)-bupropion and pure(−)-bupropion have been disclosed (Castaldi G, et al., J. Org. Chem.,1987, 52:3018, Musso et al., 1993, Chirality 5: 495-500).

A preferred embodiment of the invention envisages the combined treatmentof subjects for aid in smoking cessation or relapse prevention byvaccination using nicotine-VLP conjugates, preferentially nicotine-Qbconjugates, and administering bupropion, preferably bupropionhydrochloride. The amount of bupropion to be administered is formulatedso as to provide a initial dose of about 150 mg per day for 6 days whichis then followed by a dose of 300 mg per day.

Nortriptyline is used to treat depressions and has also been shown to beactive in aiding smoking cessation (da Costa et al., 2002, Chest, 122,403-408). Methods to produce nortryptyline are known to those skilled inthe art. A preferred embodiment of the invention envisages the combinedtreatment of subjects for aid in smoking cessation or relapse preventionby vaccination using nicotine-VLP conjugates, preferentially nicotine-Qbconjugates, and administering nortriptyline. Nortriptyline isadministered in a dose of 10-150 mg, most preferably 75 mg per day.

Additional anti-depressants contemplated for combination withvaccination include: doxepin, fluoxetine, desipramine, clomipramine,imipramine, amitriptyline, trimipramine, fluvoxamine, proxetine,sertraline, phenelzine, tranylcypromine, amoxapine, maprotiline,trazodone, venlafaxine, mirtrazapine, their pharmaceutically activesalts or their optical isomers.

Nicotine receptor agonists and antagonists attenuate the reward receivedby tobacco usage by blocking the receptors.

Varenicline tartrate is a further selective nicotinic receptormodulator. Varenicline tartrate(7,8,9,10-tetrahydro-6,10-methano-6H-pyrazino[2,3-h][3]benzazepine(2R,3R)-2,3-dihydroxybutanedionate)reduces severity of nicotine withdrawal symptoms. Its synthesis has beendescribed in WO 01/62736. A preferred embodiment of the inventionenvisages the combined treatment of subjects for aid in smokingcessation or relapse prevention by vaccination using nicotine-VLPconjugates, preferentially nicotine-Qb conjugates, and administeringvarenicline, preferably varenicline tartrate. The dose of vareniclinetartrate administered is 1 mg twice daily.

(5aS,8S,10aR)-5a,6,9,10-tetrahydro,7H,11H-8,10a-methanopyrido[2′,3′:5,6]pyrano-[2,3-d]azepine(SSR591813) is a compound that binds with high affinity alpha4beta2nicotinic acetylcholine receptor (nAChR) subtypes. The synthesis ofderivatives is described in U.S. Pat. No. 6,538,003. A preferredembodiment of the invention envisages the combined treatment of subjectsfor aid in smoking cessation or relapse prevention by vaccination usingnicotine-VLP conjugates, preferentially nicotine-Qb conjugates, andadministering SSR591813. The dose of SSR591813 is formulated to providea dose between 1 mg and 500 mg daily.

In a preferred embodiment of the invention the nicotine receptorantagonist mecamylamine hydrochloride or an pharmaceutically acceptablesalt thereof is given to subjects for aid in smoking cessation orrelapse prevention in combination with vaccination using nicotine-VLPconjugates, preferentially nicotine-Qb conjugates. Mecamylaminehydrochloride has been shown to block many of the physiological,behavioral and reinforcing effects of nicotine. Mecamylaminehydrochloride is formulated to provide a dose of about 1 mg to about 25mg per day.

Further specific nicotine antagonists include amantadine, pempidine,dihydro-beta-erthyroidine, hexamethonium, erysodine, chlorisondamine,trimethaphan camsylate, tubocurarine chloride, d-tubocurarine, theirpharmaceutically acceptable salts or their optical isomers.

Central cannabinoid receptor antagonists are also used to help quittingsmoking. Such a cannabinoid antagonist isN-piperidino-5-(4-chloro-phenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide,referred to below as rimonabant. Its synthesis and pharmaceuticalcompositions containing the same are disclosed in patent applicationsEP-576,357, EP-656,354, WO 96/02248 and WO 03/040105. The efficacy ofrimonabant has been described by Cohen et al. (Behav Pharmacol. 2002,13, 451-63).

A preferred embodiment of the invention envisages the combined treatmentof subjects for aid in smoking cessation or relapse prevention byvaccination using nicotine-VLP conjugates, preferentially nicotine-Qbconjugates, and administering rimonabant. The amount of rimonabant to beadministered is formulated so as to provide a dose of 5 to 40 mg perday, preferably 20 mg/day.

In a further embodiment opoid antagonists such as naltrexone can be usedin combination with vaccination against nicotine. The use of naltrexoneand related compounds in smoking cessation are described in U.S. Pat.No. 6,004,970. Typical doses vary between 12.5 mg and 150 mg.

Anxiolytics have also been administered to treat nicotine withdrawal.Anxiolytics counter the mild anxiety symptoms that occur during smokingcessation treatment, or the treatment of alcoholism and other substanceabuse. The anxiolytic isovaleramide has been recommended for the use insmoking cessation (Baladrin et al., WO 94/28888). Further anxiolyticscomprise buspirone, hydroxyzine and meprobamate. Buspirone isadministered in a dosage range of about 5 mg to 60 mg per day.

Monoamine oxidase inhibitors have been described for treatment of drugwithdrawal symptoms (WO 92/21333 and WO 01/12176). Reversible selectiveinhibitors of monoamine oxidase A, reversible selective inhibitors ofmonoamine oxidase B or reversible mixed inhibitors of monoamine oxidaseA and B can have activity in reducing withdrawal symptoms. Amongreversible monoamine oxidase A inhibitors befloxatone, moclobemide,brofaromine, phenoxathine, esuprone, befol, RS 8359 (Sankyo), T794(Tanabe), KP 9 (Krenitsky USA), E 2011(Eisei), toloxatone, pirlindole,amiflavine, sercloremine and bazinaprine may be cited. These compoundsare known and their preparations are described in the art. Amongreversible selective inhibitors of monoamine oxidase B, lazabemide,milacemide, caroxazone, IFO, deprenyl, AGN-1135, MDL72145 and J-508 maybe cited. The use of befloxatone or3-[4-(4,4,4-trifluoro-3R-hydroxybutoxy)phenyl]5(R)-methoxymethyl-2-oxazolidinonefor treatment of obesity has been described in WO 01/12176. The use ofthe deprenyl isomer selegeline has been described in WO92/21333.

A further compound useful in smoking cessation is clonidine (Gourlay etal., Cochrane Library 2003, 2. A preferred embodiment of the inventionenvisages the combined treatment of subjects for aid in smokingcessation or relapse prevention by vaccination using nicotine-VLPconjugates, preferentially nicotine-Qb conjugates, and administeringclonidine, perferably clonidine hydrochloride.

A further compound useful in smoking cessation is sibutramine.Sibutramine has received FDA approval to help people lose weight and itinhibits serotonin and norepinephrine reuptake. Preferably, sibutramineis given in the hydrochloride monohydrate form. Dose administered is 1to 20 mg daily, preferably 10 or 15 mg daily. A preferred embodiment ofthe invention envisages the combined treatment of subjects for aid insmoking cessation or relapse prevention by vaccination usingnicotine-VLP conjugates, preferentially nicotine-Qb conjugates, andadministering sibutramine, preferably sibutramine hydrochloride.

All drug mentioned above may be given orally as a tablet or gel capsuleor as a transdermal patches. Formulations of tablets, gel capsules andtransdermal patches are described in the art.

Smoking cessation has also been treated with a combination ofantidepressants and anxiolytics (Glazer, U.S. Pat. No. 4,788,189 or acombination of nicotine receptor antagonists and an antidepressant oranti-anxiety drug (Cary, WO 99/17803).

Further embodiments of the invention include immune molecules producedby immunization with compositions of the invention. Immune moleculesinclude antibodies and T-cell receptors. Such immune molecules may beuseful in a vaccinated individual for binding to target haptens. Immunemolecules may also be useful when transferred to another individual notimmunized against compositions of the invention, thereby to “passively”transfer immunity. In one embodiment, the immune molecule is anantibody. A monoclonal antibody suitable for binding a toxin, hormone ordrug may be transferred into an individual to achieve therapy orprophylaxis. Antibodies against nicotine and other addictive drugs mayprovide tempory alleviation of addictive behaviour. In otherembodiments, antibodies may be administered to an individual at risk ofpoisoning, or who has been acutely exposed to a toxic agent.

In another embodiment, antibodies are transferred to an individual withimmune difficiencies such as observed with cyclosporin or otherimmunosuppressive drugs, or with acquired immunodeficiency disorderse.g. HIV infection. HIV infection frequently co-occurs with addiction todrugs of abuse, particularly injectable drugs, and addiction may be anunderlying cause leading to behaviors that increase the risk ofindividual acquiring HIV infection (e.g. sharing needles, prostitution).Thus, treatment of addictive behaviour is beneficial to preventing thetransmission of HIV into uninfected individuals of the population.

In embodiments utilizing passive immunization, a pool of human donors isimmunized with conjugates of the invention using optimal immunizationregimens, as determined empirically. At various times, donors are bledby venipuncture and the titers of anti-cocaine antibody are assayed byELISA. Hyperimmune plasma from multiple donors is pooled and the IgGfraction is isolated by cold alcohol fractionation. The antibodypreparation is buffered, stabilized, preserved and standardized asneeded for hyperimmune antibody preparations for human use. The level ofanti-hapten antibody is standardized by ELISA or other antibody-basedassay.

An appropriate dose of purified antibody is administered to patientsintramuscularly, subcutaneously or intravenously. In one embodiment, theantibodies are administered with conjugate vaccine, at a differentanatomical site in order to elicit active immunity. The appropriate doseis determined by assaying serum levels of recipients in a trail patientpopulation by ELISA or other antibody-based assay at 24 hours or otherappropriate time point after injection of the hyperimmune antibodypreparation and/or assaying the effectiveness of different doses ininhibiting the effects of the hapten.

The passively transferred immune globulin inhibits the hormone, toxin ordrug effects in the patients. The use of human donors, polyclonalantibody, and the large number of donors in the donor pool limits thechance of immune response by the patients to the transferred antibody.

Other embodiments of the invention include processes for the productionof the compositions of the invention and methods of medical treatmentusing said compositions. Diverse approaches for the treatment ofaddiction are suitable as co-therapies in preventing relapse, includingpsychiatric, social and legal remedies. Pharmacologic agents useful inco-treatment of addiction include desipramine, buprenorphine, naloxone,haloperidol, chlorproazine, bromocriptine, ibogaine, mazindol,antidepressants and others that will be apparent to the ordinarilyskilled artisan.

Kits

The invention also embodies the use of antibodies produced byimmunization with compositions of the invention in kits for thedetection of haptens in immunoassays (eg ELISA). In a relatedembodiment, repetitive ordered hapten arrays can be useful for thedetection of antibodies against haptens in binding assays.

In some specific embodiments, the compositions of the present inventionmay be assembled into kits for use in detection in assays or industrialsettings, in diagnosis or detection of diseases, conditions ordisorders. Such kits according to the present invention may comprise atleast one container containing one or more of the above-describedconjugates or compositions, including hapten-core particle conjugatesand immune molecules directed against such conjugates. Alternative kitsof the invention may comprise one or more antibodies of the inventionproduced by the methods of the invention or by immunization methodsfamiliar to the ordinarily skilled artisan using the conjugates andcompositions of the present invention. The kits of the invention mayoptionally further comprise at least one additional container which maycontain, for example, one or more antigens, one or more haptens, one ormore core particles, one or more conjugates/compositions of theinvention, one or more pharmaceutically acceptable carriers orexcipients, one or more buffers, one or more proteins, one or morenucleic acid molecules, and the like

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody, produced by amethod of the invention, preferably a purified antibody, in one or morecontainers. In a specific embodiment, the kits of the present inventioncontain a substantially isolated hapten which is specificallyimmunoreactive with an antibody included in the kit. Preferably, thekits of the present invention further comprise a control antibody whichdoes not react with the hapten of interest. In another specificembodiment, the kits of the present invention contain a means fordetecting the binding of an antibody to a hapten of interest (e.g., theantibody may be conjugated to a detectable substrate such as afluorescent compound, an enzymatic substrate, a radioactive compound ora luminescent compound, or a second antibody which recognizes the firstantibody may be conjugated to a detectable substrate).

In another specific embodiment of the present invention, the kit is adiagnostic kit for use in screening serum containing antibodies specificagainst Nicotine. Such a kit includes antibodies of IgA, IgE, IgG andIgG subclasses directed against nicotine and obtained by theimmunization of a human with nicotine-Qβ VLP conjugates of the presentinvention. Such a kit includes a control antibody that does not reactwith nicotine, and substantially isolated nicotine, cotinine andnornicotine haptens, and purified core particle free of hapten. Further,such a kit includes means for detecting the binding of said antibody tonicotine hapten (e.g., the antibody may be conjugated to a fluorescentcompound such as fluorescein or rhodamine which can be detected by flowcytometry, or HRP for use in an ELISA). In one specific embodiments, thekit may include a nicotine attached to a solid support. The inventionembodies the use of such a kit, where the titre of differentimmunoglobulin classes and subclasses are determined in an ELISA. Theanti nicotine IgA, IgE and IgG antibodies provided in the kit serve ascontrols to assess the relative titre of antibodies in the patientserum. After binding of the antibody of the serum and the kit withnicotine hapten, and removing unbound serum components by washing, theantibodies are reacted with with antibodies specific for immunoglobulinsubtypes that are conjugated to reporter molecules. After a furtherwashing step, to remove unbound labeled antibody, and the amount ofreporter associated with the solid phase is determined in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(Sigma, St. Louis, Mo.).

Thus, by the use of the above kits, the invention provides a method formonitoring the progress of immunization against nicotine. An immunizedperson will be monitored during the course of immunization for IgG andIgA antibodies against nicotine, and for the lack IgE antibodies againstnicotine that would indicate the development of an allergic reaction. Ifthe immune response is primarily against the core particle rather thanthe hapten, an alternative nicotine conjugate will be utilized, with adifferent core partile and, in one embodiment, a different hapten.

In one embodiment a kit includes a solid support to which anNicotine-core particle conjugate is attached. In this embodiment,binding of antibody in the serum of an individual to the antigenpresented on the core particle can be detected by binding of areporter-labeled antibody.

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Thus, the invention provides an assay systems or kits for carrying out adiagnostic method. The kit generally includes bound recombinant antigenand a reporter-labeled antibody for detecting bound anti-antigenantibody. Other suitable kits of the present invention are understood tothose of ordinary skill in the art.

It will be understood by one of ordinary skill in the relevant arts thatother suitable modifications and adaptations to the methods andapplications described herein are readily apparent and may be madewithout departing from the scope of the invention or any embodimentthereof. Having now described the present invention in detail, the samewill be more clearly understood by reference to the following examples,which are included herewith for purposes of illustration only and arenot intended to be limiting of the invention.

Examples Example 1 Coupling Procedure for Nicotine-Qβ Conjugate

A nicotine derivate suitable for coupling to VLPs was synthesizedaccording Langone et al. (1982, supra). Trans-4′-carboxycotinine isavailable from commercial sources. The methylester oftrans-4′-carboxycotinine is produced by reactingtrans-4′-carboxycotinine with methanolic sulfuric acid. The solution isneutralized with sodium bicarbonate, extracted with chloroform,concentrated on a rotary evaporator and recrystallized fromether-acetone. Reduction of the methyl ester with lithium aluminiumhydride in ether then produces trans-3′-hydroxymethylnicotine. TheO′-succinyl-hydroxymethylnicotine is then produced by the addition ofsuccinic anhydride in benzene. The solution is concentrated on a rotaryevaporator. Activation of the carboxyl group is subsequently achieved byaddition of EDC (1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide) andN-hydroxysuccinimide (NHS) resulting in the N-hydroxysuccinimide esterof O′-succinyl-hydroxymethylnicotine (in the following abbreviated as“Suc-Nic”).

Qβ CP VLPs (SEQ ID NO: 3) were dialysed against Hepes-buffered salineHBS (50 mM Hepes, 150 mM NaCl, pH 8.0). The nicotine derivative Suc-Nicwas dissolved in HBS at a concentration of 121 mM. It was added to a QβCP VLP solution (0.14 mM) at 1×, 5×, 50×, 100× and 500× molar excess andincubated at room temperature for 2 h on a shaker. The reaction solutionwas then dialysed against HBS, pH 8.0 (cut off 10000 Da), flash-frozenin liquid nitrogen and stored at −80° C. The nicotine derivative suc-nicreacts with lysines on the surface of Qβ under formation of an amidbond. The resulting covalent conjugate is termed herein “Nic-Qβ”.

SDS-PAGE analysis showed with increasing molar excess of Suc-Nic a shiftof the Qβ monomer band to higher molecular weights (FIG. 1A). Thepresence of nicotine in the coupling product was confirmed by awesternblot using an anti-nicotine antiserum. While uncoupled Qβ controland Qβ coupled to nicotine at a 1× and 5× excess did not show ananti-nicotine reactive band, the bands at 50×, 100× and 500× clearlydemonstrated covalent coupling of nicotine to Qβ (FIG. 1B). This wasconfirmed by an ELISA with nicotine-BSA coated on the wells anddetection with an anti-nicotine antiserum. A higher absorbance wasreached when Qβ coupled with 500 fold excess nicotine was used comparedto a vacccine produced with an 50 fold excess.

Example 2 Immunization of Mice with Nic-Qβ and Measurement ofAnti-Nicotine Antibody Titers

A. Immunization of Mice

10 week-old female Balb/c mice were vaccinated twice with 30 μg of thenicotine-Qβ (Nic-Qβ) resulting from the coupling using 500× excess ofSuc-Nic. The vaccine was diluted in sterile PBS and given intranasallyor injected subcutaneously with or without the addition of Alum (Imject,Pierce). 14 days after the first immunization the mice were boosted(Table I). On day 29 the nicotine-specific antibody titers in serum weredetermined by ELISA.

TABLE I Immunization scheme of mice: B. ELISA. Day 0 Day 14 AmountAmount Day 29 No. of animals vaccine (μg) (μg) Bled 3 Nic-Qβ s.c. 30 30Bled 3 Nic-Qβ s.c. & Alum 30 30 Bled 3 Nic-Qβ intranasal 30 30 Bled

Sera were analyzed in a nicotine-specific ELISA: Microtiter plates(Maxisorp, Nunc) were coated overnight with 5 μg/ml nicotine coupled toBSA (NAB03) in coating buffer (pH 9.6). After washing and blocking with2% BSA in PBS, sera were added at different dilutions in 2% BSA/1%FCS/PBS. After 2 hours incubation at room temperature the plates werewashed (0.05% Tween 20/PBS) and HRPO-labeled antibodies specific formouse antibody subclasses were added. After 1 hour incubation the plateswere washed and the color substrate OPD in citric acid buffer was added.After 5 minutes the color reaction was stopped with 5% H₂SO₄.

Optical densities at 450 nm were read in an ELISA Reader (Benchmark,Becton Dickinson). For the detection of IgE, sera were pre-incubated inEppendorf tubes with Protein G beads (Pierce) for 30 min on a shakerbefore adding to the ELISA plate.

The Nic-Qβ vaccine induced nicotine-specific IgG antibodies (FIG. 3A).The ELISA titers were calculated for the total IgG response (FIG. 3B,Table II). The ELISA titer was defined as the dilution of the serumwhich gives a half-maximal optical density signal (OD 50%)) in theELISA. For the subcutaneous route with Alum, the average IgG titersobtained with Nic-Qβ were13228. For the intranasal route, nicotine-Qβtiters were 38052.

IgG subtypes and IgE were also measured by ELISA and titers determined(FIG. 3, FIG. 4, Table II). No significant IgE response above background(preimmune serum) could be detected for any of the conditions tested.The ratio of IgG2a to IgG1 antibody titers is indicative for a Th1mediated immune response. A ratio of 2.1 was measured for the miceimmunized subcutanously with Nic-Qβ in the absence of Alum, and was evenenhanced to 2.6 when applied intranasally. As expected Alum drove theimmune response towards a more Th2 type response and resulted in a ratioof 0.4. Notably, the Nic-Qβ vaccines also induced high IgG2b and IgG3titers. A significant amount of anti-nicotine IgA antibodies could befound in serum (FIG. 5) which are indicative for the presence of IgA inmucosal surfaces.

The high nicotine titres upon intranasal immunization are especiallynoteworthy.

TABLE II Nicotine-specific antibody titers in vaccinated mice Titerswere calculated as the dilution of serum that gives half-maximal opticaldensity in the ELISA. Average titers of 3 mice each are given. IgG2bIgG3 Vaccine IgG titer IgG1 titer IgG2a titer titer titer Nic-Qβ s.c.13228 672 1386 515 2030 Nic-Qβ alum s.c. 93762 9642 10016 14977 19701Nic-Qβ intranasal 38052 2845 7493 3617 6107

Example 3 Evaluation of Nicotine Distribution in Plasma and Brain inRats

Groups of rats are immunized with the nicotine-VLP vaccine, boosted atday 21. One group receives a second boost at day 35. Seven to 10 daysafter the last boost rats are anestethized and catheters are placed inthe femoral artery and vein for sampling and the jugular vein of theother leg for nicotine adminstration. Nicotine 0.03 mg/kg containing 3microCi 3H-(−)-nicotine is infused in 1 ml/kg 0.9% saline via thejugular vein over 10 s. The radiolabel is added to permit estimation ofnicotine concentrations from very small volumes of blood. This thepossible because metabolism of nicotine to cotinine over the first 90 safter nicotine administration in rats negligible. Blood (0.3 ml) wasremoved from both the femoral artery and and vein catheers every 15 s upto 90 s, centrifuged immediately and serum separated for assay. Rats arekilled at 3 min by decapitation, the brain is removed quickly, rinsedwith water and stored at −20° C. until assayed. For measurement of3H-nicotine concentration in serum, 100 ul serum is mixed with liquidscintillation fluid. Brain samples were digested in 5 vol NaOH prior toextraction and analysed after addition of scintillation fluid.

Nicotine-specific antibodies induced by the vaccination are capable ofbinding 3H-nicotine in serum and inhibit or lower its diffusion into thebrain. Accordingly, a decreased concentration of brain nicotine and anincreased concentration of plasma nicotine are measured.

Example 4 Chemical Coupling of Nicotine Hapten to HbcAg-Lys

O-succinyl-hydroxymethylnicotine is prepared as described in Example 1,and incubated with EDC and NHS to yield the activatedN-hydroxysuccinamide ester (Suc-Nic). Purified HbcAg-Lys VLP is preparedas described in copending U.S. patent application Ser. No. 10/050,902.Suc-Nic solution in HBS is added at 1×, 5×, 50×, 100× and 500× molarexcess to a 95% pure solution of HBcAg-Lys particles (2 mg/ml) andincubated for 2 h. at room temperature. After completion of thereaction, the mixture is dialyzed overnight against HBS, pH 8.0, flashfrozen in liquid nitrogen and stored at −80° C. Reaction is monitored bySDS-PAGE analysis and western blot with antinicotine antiserum. Nicotinedecorated particles are injected into rodents to induce immune responsesagainst nicotine.

Example 5 Chemical Coupling of Nicotine Hapten to Type-1 Pili ofEscherichia coli

Type I pili are prepared from E. coli strain W3110 transformed with thevector pFIMAICDFGK, and purified by ultracentrifugation, as described incommonly owned, co-pending U.S. patent application Ser. No. 10/050,902,filed Jan. 18, 2002, the disclosure of which is incorporated herein byreference in its entirety. Activated hapten Suc-Nic in HBS are added at1×, 5×, 50×, 100× and 500× molar excess to a 95% pure solution of type Ipili particles (2 mg/ml) and incubated for 2 h. at room temperature.After completion of the reaction, the mixture is dialyzed against HBS,pH 8.0, flash frozen in liquid nitrogen and stored at −80° C. Reactionis monitored by SDS-PAGE analysis and western blot with antinicotineantiserum. Nicotine decorated particles are injected into rodents toinduce immune responses against nicotine.

Example 6 Synthesis of Multi-hapten Vaccine Suitable for Treatment ofNicotine Addiction

A vaccine against nicotine addiction designed to target multipleepitopes of nicotine and also the pharmaceutically active metabolitescotinine and nornicotine is prepared. Individual 120 mM solutions in HBSof 6-(carboxymethylureido)-(±)-nicotine (CMUNic),trans-3′-aminomethylnicotine succinate,O-succinyl-3′-hydroxymethyl-nicotine, Trans-4′-carboxycotinine,N-[1-oxo-6-[(25)-2-(3-pyridyl)-1-pyrrolidinyl]hexyl]-β-alanine,4-oxo-4-[[6-[(5S)-2-oxo-5-(3-pyridinyl)-1-pyrrolidinyl]]hexyl]amino]-butanoicacid, (2S)-2-(3-pyridinyl)-1-pyrrolidinebutanoic acid phenylmethylester, (2R)-2-(3-pyridinyl)-1-pyrrolidinebutanoic acid phenylmethylester, Cotinine 4′-carboxylic acid, N-succinyl-6-amino-(.±.)-nicotine;6-(.sigma.-aminocapramido)-(.±.)-nicotine- and6-(.sigma.-aminocapramido)-(.±.)-nicotine-conjugates; succinylated3′,4′, and 5′ aminomethylnicotine, 5 and 6 aminonicotine and 3′,4′, and5′ acetyl derivatives of acetyl nicotine. The solutions are mixed withEDC and NHS to form activated forms which are added, in separatereactions, at 10-100 molar excess to Qβ VLP as described elsewhere.

Individual solutions of S-1-(b-aminoethyl)nicotinium chloridedihydrochloride and S-1-(b-aminoethyl)cotinium chloride hydrochloridesolutions are coupled to Qβ VLP with1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimidemetho-p-toluenesulfonate.

From this selection of conjugates, eight of the nicotine hapten Qβ VLPconjugates, a cotinine Qβ VLP conjugate and a nornicotine conjugate QβVLP are then admixed to form a vaccine composition, which is used tovaccinate individuals. After 2 doses, individuals are then boosted 3times with parallel haptens coupled to HBc-Lys VLP conjugates.

Example 7 Synthesis of Cocaine VLP-Hapten Conjugate

A solution of norcocaine hydrochloride (1 g, 3.07 mmol), triethylamine(0.86 ml, 6.14 mmol) in methylene chloride (20 ml) is treated withsuccinic anhydride (614 mg, 6.14 mmol) and the mixture heated at45.degree. C. overnight, as described in U.S. Pat. No. 5,876,727. Thesolvents are removed under reduced pressure and the residue purifiedusing silica gel flash chromatography (2:1 chloroform:methanol as theeluent). This gives succinylated norcocaine (1.0 g, 84%) as a thicksyrup(3.beta.-(Benzoyloxy)-8-succinoyl-8-azabicyclo[3.2.1]octane-2.beta.-carboxylicacid methyl ester). To a solution of the acid (14 mg, 0.036 mmol) indistilled water (1 ml) at 0.degree. C., EDC (10.4 mg, 0.055 mmol) wasadded. After 5 minutes a solution of Qβ VLP in PBS (1 ml) is addeddropwise and the mixture is allowed to warm to ambient temperatureovernight. The conjugate is purified by dialysis against PBS and thedegree of conjugation analyzed by mass spectral analysis. The resultantconjugate is used to immunize individuals.

Having now fully described the present invention in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious to one of ordinary skill in the art that the same can beperformed by modifying or changing the invention within a wide andequivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any specific embodimentthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

Exmaple 8 Evaluation of Nicotine Distribution in Plasma and Brain ofMice

Groups of 4 to 5 mice were immunized with 60 ug of the nicotine-VLPvaccine produced as described in EXAMPLE 1 and were boosted at day 35and day 63 with the same amount of vaccine. Fourteen days after the lastboost mice were injected i.v. at the base of the tail with a solutioncontaining 750 ng (−)-nicotine hydrogen tartrate with 5 microCi3H-(−)-nicotine. The amount of nicotine corresponds to 0.03 mg/kg whichis equivalent to the nicotine uptake of 2 cigarettes by a smoker. Theradiolabel was added to permit estimation of nicotine concentrationsfrom very small volumes of blood. After five minutes mice weresacrificed by CO₂. Blood was removed by punctation of the heart andserum was prepared. Brains was immediately dissected, cleaned fromadhering blood and their weights measured. For measurement of3H-nicotine concentration in serum, 50 ul serum is mixed with liquidscintillation fluid. Brain samples were completely dissolved in 2 mlTissue Solubilizer (Serva) and analysed after addition of scintillationfluid. From the radioactivities nicotine concentrations in serum andbrain were calculated (FIG. 7).

Nicotine-specific antibodies induced by the vaccination were capable ofbinding 3H-nicotine in serum and inhibit or lower its diffusion into thebrain. Accordingly, a decreased concentration of brain nicotine and anincreased concentration of plasma nicotine were measured. TheNicotine-VLP vaccine was able to inhibit the nicotine uptake in brain by56% in the absence of Alum and by 68% in the presence of Alum (FIG. 7).

Further immunization schedules, such as immunization at day 0 and andboosting at day 14 also yielded in antibodies levels that were able tosignificantly reduce nicotine uptake into brain. In general, highanti-nicotine antibody titers correlated with a higher efficacy of thevaccination.

Example 9 Cloning of the AP205 Coat Protein Gene

The cDNA of AP205 coat protein (CP) (SEQ ID NO: 90) was assembled fromtwo cDNA fragments generated from phage AP205 RNA by using a reversetranscription-PCR technique and cloning in the commercial plasmid pCR4-TOPO for sequencing. Reverse transcription techniques are well knownto those of ordinary skill in the relevant art. The first fragment,contained in plasmid p205-246, contained 269 nucleotides upstream of theCP sequence and 74 nucleotides coding for the first 24 N-terminal aminoacids of the CP. The second fragment, contained in plasmid p205-262,contained 364 nucleotides coding for amino acids12-131of CP and anadditional 162 nucleotides downstream of the CP sequence. Both p205-246and p205-262 were a generous gift from J. Klovins.

The plasmid 283.-58 was designed by two-step PCR, in order to fuse bothCP fragments from plasmids p205-246 and p205-262 in one full-length CPsequence.

An upstream primer p1.44 containing the NcoI site for cloning intoplasmid pQb185, or p1.45 containing the XbaI site for cloning intoplasmid pQb10, and a downstream primer p1.46 containing the HindIIIrestriction site were used (recognition sequence of the restrictionenzyme underlined):

(SEQ ID NO: 5) p1.44 5′-NNCC ATG GCA AAT AAG CCA ATG CAA CCG-3′ (SEQ IDNO: 20) p1.45 5′-NNTCTAGAATTTTCTGCGCACCCATCCCGG-3′ (SEQ ID NO: 21) p1.465′-NNAAGC TTA AGC AGT AGT ATC AGA CGA TAC G-3′

Two additional primers, p1.47, annealing at the 5′ end of the fragmentcontained in p205-262, and p1.48, annealing at the 3′ end of thefragment contained in plasmid p205-246 were used to amplify thefragments in the first PCR. Primers p1.47 and p1.48 are complementary toeach other.

(SEQ ID NO: 22) p1.47: 5′-GAGTGATCCAACTCGTTTATCAACTACATTT-TCAGCAAGTCTG-3′ (SEQ ID NO: 23) p1.48:5′-CAGACTTGCTGAAAATGTAGTTGATAAACGA- GTTGGATCACTC-3′

In the first two PCR reactions, two fragments were generated. The firstfragment was generated with primers p1.45 and p1.48 and templatep205-246. The second fragment was generated with primers p1.47 andp1.46, and template p205-262. Both fragments were used as templates forthe second PCR reaction, a splice-overlap extension, with the primercombination p1.45 and p1.46 or p1.44 and p1.46. The product of the twosecond-step PCR reactions were digested with XbaI or NcoI respectively,and HindIII, and cloned with the same restriction sites into pQb10 orpQb185 respectively, two pGEM-derived expression vectors under thecontrol of E.coli tryptophan operon promoter.

Two plasmids were obtained, pAP283-58 (SEQ ID NO: 15), containing thegene coding for wt AP205 CP (SEQ ID NO: 14) in pQb10, and pAP281-32 (SEQID NO: 19) with mutation Pro5→Thr (SEQ ID NO: 18), in pQb185. The coatprotein sequences were verified by DNA sequencing. PAP283-58 contains 49nucleotides upstream of the ATG codon of the CP, downstream of the XbaIsite, and contains the putative original ribosomal binding site of thecoat protein mRNA.

Example 10 Expression and Purification of Recombinant AP205 VLP A.Expression of Recombinant AP205 VLP

E. coli JM109 was transformed with plasmid pAP283-58. 5 ml of LB liquidmedium with 20 μg/ml ampicillin were inoculated with a single colony,and incubated at 37° C. for 16-24 h without shaking.

The prepared inoculum was diluted 1:100 in 100-300 ml of LB medium,containing 20 μg/ml ampicillin and incubated at 37° C. overnight withoutshaking. The resulting second inoculum was diluted 1:50 in 2TY medium,containing 0.2% glucose and phosphate for buffering, and incubated at37° C. overnight on a shaker. Cells were harvested by centrifugation andfrozen at −80° C.

B. Purification of Recombinant AP205 VLP

Solutions and buffers:

-   1. Lysis buffer    -   50 mM Tris-HCl pH 8.0 with 5 mM EDTA , 0.1% tritonX100 and PMSF        at 5 micrograms per ml.-   2. SAS    -   Saturated ammonium sulphate in water-   3. Buffer NET.    -   20 mM Tris-HCl, pH 7.8 with 5 mM EDTA and 150 mM NaCl.-   4. PEG    -   40% (w/v) polyethylenglycol 6000 in NET

Lysis:

Frozen cells were resuspended in lysis buffer at 2 ml/g cells. Themixture was sonicated with 22 kH five times for 15 seconds, withintervals of lmin to cool the solution on ice. The lysate was thencentrifuged for 20 minutes at 12 000 rpm, using a F34-6-38 rotor(Ependorf). The centrifugation steps described below were all performedusing the same rotor, except otherwise stated. The supernatant wasstored at 4° C., while cell debris were washed twice with lysis buffer.After centrifugation, the supernatants of the lysate and wash fractionswere pooled.

Ammonium-sulphate precipitation can be further used to purify AP205 VLP.In a first step, a concentration of ammonium-sulphate at which AP205 VLPdoes not precipitate is chosen. The resulting pellet is discarded. Inthe next step, an ammonium sulphate concentration at which AP205 VLPquantitatively precipitates is selected, and AP205 VLP is isolated fromthe pellet of this precipitation step by centrifugation (14 000 rpm, for20 min). The obtained pellet is solubilised in NET buffer.

Chromatography:

The capsid protein from the pooled supernatants was loaded on aSepharose 4B column (2.8×70 cm), and eluted with NET buffer, at 4ml/hour/fraction. Fractions 28-40 were collected, and precipitated withammonium sulphate at 60% saturation. The fractions were analyzed bySDS-PAGE and Western Blot with an antiserum specific for AP205 prior toprecipitation. The pellet isolated by centrifugation was resolubilizedin NET buffer, and loaded on a Sepharose 2B column (2.3×65 cm), elutedat 3 ml/h/fraction. Fractions were analysed by SDS-PAGE, and fractions44-50 were collected, pooled and precipitated with ammonium sulphate at60% saturation. The pellet isolated by centrifugation was resolubilizedin NET buffer, and purified on a Sepharose 6B column (2.5×47 cm), elutedat 3 ml/hour/fraction. The fractions were analysed by SDS-PAGE.Fractions 23-27 were collected, the salt concentration adjusted to 0.5M, and precipitated with PEG 6000, added from a 40% stock in water andto a final concentration of 13.3%. The pellet isolated by centrifugationwas resolubilized in NET buffer, and loaded on the same Sepharose 2Bcolumn as above, eluted in the same manner. Fractions 43-53 werecollected, and precipitated with ammonium sulphate at a saturation of60%. The pellet isolated by centrifugation was resolubilized in water,and the obtained protein solution was extensively dialyzed againstwater. About 10 mg of purified protein per gram of cells could beisolated. Examination of the virus-like particles in Electron microscopyshowed that they were identical to the phage particles.

1. A nicotine hapten-carrier conjugate comprising: (a) a virus-likeparticle of a RNA-phage carrier comprising at least one first attachmentsite, and (b) at least one nicotine hapten with at least one secondattachment site; wherein said second attachment site is associatedthrough at least one covalent bond to said first attachment site so asto form an ordered and repetitive hapten-carrier conjugate. 2.(canceled)
 3. The conjugate of claim 1, wherein said virus-like particleis a recombinant virus-like particle. 4-9. (canceled)
 10. The conjugateof claim 3 wherein said virus-like particle comprises recombinantproteins, or fragments thereof, of RNA-phage Qβ. 11-12. (canceled) 13.The conjugate of claim 3, wherein said virus-like particle comprisesrecombinant proteins, or fragments thereof, of a RNA-phage, and whereinsaid recombinant proteins consist of coat proteins of RNA phages. 14-20.(canceled)
 21. The conjugate of claim 10, wherein said recombinantproteins comprise coat proteins having an amino acid sequence as setforth in SEQ ID NO:3, or a mixture of coat proteins having amino acidsequences of SEQ ID NO: 4, or mutants thereof, and of SEQ ID NO:3. 22.The conjugate of claim 3, wherein said virus-like particle essentiallyconsisting of coat proteins having an amino acid sequence of SEQ IDNO:3, or essentially consisting of a mixture of coat proteins havingamino acid sequences of SEQ ID NO: 4, or mutants thereof, and of SEQ IDNO:3. 23-29. (canceled)
 30. The conjugate of claim 1, wherein said atleast one first attachment site is a lysine residue.
 31. The conjugateof claim 1, wherein said second attachment site is associated to saidfirst attachment site through at least one non-peptide covalent bond.32. (canceled)
 33. The hapten-carrier conjugate of claim 1, wherein saidconjugate is suitable for eliciting an immune response against nicotine.34-37. (canceled)
 38. The conjugate of claim 33, wherein the conjugateis formed from starting materials selected from the group consisting of:(a) 6-(carboxymethylureido)-(±)-nicotine (CMUNic); (b)trans-3′-aminomethylnicotine succinate; (c)O-succinyl-3′-hydroxymethyl-nicotine; (d) Trans-4′-carboxycotinine; (e)N-[1-oxo-6-[(25)-2-(3-pyridyl)-1-pyrrolidinyl]hexyl]-β-alanine; (f)6-(.sigma.-aminocapramido)-(±)-nicotine; (g) 3′ aminomethylnicotine; (h)4′aminomethylnicotine; (i) 5′ aminomethylnicotine; (j) 5 aminonicotine;(k) 6 aminonicotine; (l) S-1-(b-aminoethyl)nicotinium chloride; and (m)S-1-(b-aminoethyl)cotinium chloride.
 39. The conjugate of claim 33,wherein said hapten comprises the starting materialO-succinyl-3′-hydroxymethyl-nicotine.
 40. The conjugate of claim 33,wherein said conjugate comprises O-succinyl-3′-hydroxymethyl-nicotineconjugated to Qβ virus like particle.
 41. The conjugate of claim 33,wherein said hapten is formed from the starting materialO-succinyl-3′-hydroxymethyl-nicotine.
 42. The conjugate of claim 41,wherein the second attachment site contains, preferably is, an activegroup selected from the group consisting of (a) Amine; (b) Amide; (c)Carboxyl; (d) Sulfhydryl; (e) Hydroxyl; (f) Aldehyde; (g) Diazonium; (h)Alcylhalogenid; (i) Hydrazine; (j) Vinyl; (k) Maleimid; (l) Succinimide;and (m) Hydrazide.
 43. The conjugate of claim 42, wherein said secondattachment site is formed by reaction of the O-succinyl moiety of saidO-succinyl-3′-hydroxymethyl-nicotine with the first attachment site. 44.The conjugate of claim 41, wherein the second attachment site containsan amide, and wherein said second attachment site is formed by reactionof the O-succinyl moiety of said O-succinyl-3′-hydroxymethyl-nicotinewith a lysine residue being said first attachment site.
 45. (canceled)46. The conjugate of claim 33, wherein said conjugate comprisesO-succinyl-3′-hydroxymethyl-nicotine conjugated to a virus-like particleof a RNA-phage, preferably to a Qβ virus like particle, and herebypreferably to a Qβ virus like particle comprising, or preferably beingcomposed of coat proteins of .RNA-phage Qβ. 47-62. (canceled)
 63. Apharmaceutical composition comprising the conjugate of claim 33; and apharmaceutically acceptible carrier. 64-65. (canceled)
 66. A vaccinecomposition comprising the conjugate of claim
 1. 67-68. (canceled)
 69. Amethod of inducing an immune response to a drug in an animal, saidmethod comprising administering an immunologically effective amount ofthe conjugate of claim 33 to an animal and permitting said animal toproduce an immune response to said drug.
 70. The method of claim 69,wherein said conjugate is administered to said animal by a routeselected from the group consisting of: intranasally, orally,subcutaneously, transdermally, intramuscularly or intravenously.71.-115. (canceled)
 116. The conjugate of claim 1, wherein saidvirus-like particle is a virus-like particle of RNA-phage Qβ coatprotein.
 117. The conjugate of claim 116, wherein said virus-likeparticle of RNA-phage Qβ coat protein comprising a coat proteinconsisting of the amino acid sequence as set forth in SEQ ID NO:3. 118.The conjugate of claim 117, wherein said first attachment site is alysine residue and, wherein said first attachment site naturally occursin said core particle.